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ONCOKINASE FUSION POLYPEPTIDES ASSOCIATED WITH HYPERPROLIFERATIVE AND RELATED DISORDERS, NUCLEIC ACIDS ENCODING THE SAME AND METHODS FOR DETECTING AND IDENTIFYING THE SAME - Patent applicationinit();
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Abstract:

Oncokinase fusion polypeptides associated with hyperproliferative
disorders and the polynucleotides encoding for such fusion polypeptides
are provided. The fusion polypeptides have a C-terminal tyrosine kinase
domain fused to an N-terminal domain that is not normally fused to the
C-terminal tyrosine kinase domain and they possess constitutively
activated tyrosine kinase activity. Also provided are methods for
detecting and identifying the fusion polypeptides and polynucleotides and
methods of diagnosing disease conditions associated with the fusion
polypeptides and polynucleotides. In addition, screening assays for
identifying agents useful for treating disease conditions associated with
such fusion polypeptides and polynucleotides are provided. Furthermore,
methods of treating disease conditions associated with the presence of
the fusion polypeptides are provided.

Claims:

1. A fusion polypeptide comprising a C-terminal tyrosine kinase domain and
an N-terminal domain wherein said fusion polypeptide has a constitutively
activated kinase activity, is present in other than its naturally
occurring environment and is further characterized by at least one of the
following features:(a) said C-terminal domain is from a chromosome 4
encoded protein;(b) said N-terminal domain is from a chromosome 4 encoded
protein; and(c) said fusion protein is not a product of a translocation
event.

2. The fusion polypeptide according to claim 1, wherein said C-terminal
domain is from a chromosome 4 encoded protein.

3. The fusion polypeptide according to claim 1, wherein said N-terminal
domain is from a chromosome 4 encoded protein.

4. The fusion polypeptide according to claim 1, wherein said fusion
protein is not a product of a translocation event.

5. The fusion polypeptide according to claim 1, wherein said chromosome 4
tyrosine kinase domain comprises the C-terminal domain of a tyrosine
kinase chosen from PDGFRα, c-Kit and VEGFR-2.

6. The fusion polypeptide according to claim 1, wherein said tyrosine
kinase is PDGFRα.

7. The fusion polypeptide according to claim 1, wherein said N-terminal
domain is a NM--030917 domain.

8. The fusion polypeptide according to claim 1, wherein said fusion
polypeptide is a primate polypeptide.

9. The fusion polypeptide according to claim 8, wherein said primate
polypeptide is a human polypeptide.

10. The fusion polypeptide according to claim 1, wherein said fusion
polypeptide has an amino acid sequence that is substantially the same as
or identical to a sequence chosen from SEQ ID NO:01, SEQ ID NO:02; SEQ ID
NO:03 and SEQ ID NO:04.

11. The fusion polypeptide according to claim 1, wherein said fusion
polypeptide is substantially pure.

12-53. (canceled)

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001]Pursuant to 35 U.S.C. §119 (e), this application claims
priority to the filing date of the U.S. Provisional Patent Application
Ser. No. 60/402,330 filed Aug. 9, 2002 and U.S. Provisional Patent
Application Ser. No. 60/440,491 filed Jan. 16, 2003; the disclosures of
which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

[0002]1. Field of the Invention

[0003]This invention is directed to novel oncokinase fusion polypeptides
associated with hyperproliferative disorders and to the polynucleotides
that encode for such fusion polypeptides. This invention is also directed
to methods of identifying and characterizing such fusion polypeptides and
polynucleotides; to methods of diagnosing disease conditions associated
with such fusion polypeptides and polynucleotides; and to screening
assays for identifying agents useful for treating disease conditions
associated with such fusion polypeptides and polynucleotides.

[0004]2. State of the Art

[0005]An accumulation of genetic changes underlies the development and
progression of hyperproliferative disorders, such as cancer, resulting in
cells that differ from normal cells in their behavior, biochemistry,
genetics, and microscopic appearance. Mutations in DNA that cause changes
in the expression level of key proteins, or in the structures and
biological activities of proteins, are thought to be at the heart of
cancer. For example, cancer can be triggered when genes that play a
critical role in the regulation of cell growth and survival undergo
mutations that lead to their over-expression and/or activation. Such
"oncogenes" are involved in the dysregulation of growth that occurs in
cancers.

[0006]Kinases and phosphatases are enzymes involved in phosphorylation and
dephosphorylation that help regulate many cellular activities,
particularly signaling from the cell membrane to the nucleus to initiate
the cell's entrance into the cell cycle and to control other functions.
For example, phosphorylation is important in signal transduction mediated
by receptors via extracellular biological signals such as growth factors
or hormones. Many oncogenes are kinases or phosphatases, i.e. enzymes
that catalyze protein phosphorylation or dephosphorylation reactions.
Kinases and phosphatases may themselves be specifically regulated by
phosphorylation. A kinase or phosphatase can have its activity regulated
by one or more distinct kinase or phosphatases, resulting in specific
signaling cascades.

[0007]Despite a long-standing need to understand and discover methods for
regulating cells involved in various disease states, the complexity of
signal transduction pathways has been a barrier to the development of
products and processes for such regulation. Accordingly, there is a need
in the art for improved methods for detecting and modulating the activity
of genes involved in signal transduction and cell cycle regulation and
for treating diseases associated with cancer and related disease
conditions resulting from abnormal phosphorylation activity, e.g., kinase
activity.

SUMMARY OF THE INVENTION

[0008]Oncokinase, particularly tyrosine kinase, fusion polypeptides
associated with hyperproliferative disorders, as well as nucleic acids
encoding the same, are provided. A feature of the subject fusion
polypeptides is that they include a C-terminal tyrosine kinase domain
fused to an N-terminal domain that is not normally fused to the
C-terminal tyrosine kinase domain, where the subject fusion polypeptides
possess constitutively activated tyrosine kinase activity; i.e., they do
not require the presence of an exogenous factor, e.g., a growth factor,
to express their catalytic activity. The subject fusion polypeptides are
further characterized in that they include at least one of the following
features: (a) the C-terminal domain is from a chromosome 4 tyrosine
kinase; (b) the N-terminal domain is from a chromosome 4 encoded protein,
e.g., it is a NM--030917 domain; and (c) the fusion protein does not
arise from a translocation event, i.e., it does not arise from the
exchange of DNA between different chromosomes; where in certain
embodiments, two or more, including all three of, these features are
present in the subject fusion polypeptides. Also provided are methods of
identifying and characterizing the subject fusion polypeptides. Also
provided are methods of diagnosing disease conditions by detecting the
presence of the subject polypeptides/polynucleotides and/or detecting the
deletions of one or more genomic sequences, where the deletions result
from the chromosomal deletion event that gives rise to the subject
polypeptides/polynucleotides. In addition, screening assays for
identifying agents that find use in treating disease conditions
associated with the presence of the subject fusion polypeptides are
provided. Furthermore, methods of treating disease conditions associated
with the presence of the subject fusion polypeptides are provided.

[0010]FIG. 2 provides a diagram illustrating the genetic rearrangement
that gives rise to the oncogene and fusion polypeptide of the subject
invention.

[0011]FIGS. 3A and 3B provide sequence data of fusion points found in
NM--030917-PDGFRα fusion protein coding sequences found in two
different EOS patients.

DETAILED DESCRIPTION OF THE INVENTION

[0012]Oncokinase, particularly tyrosine kinase, fusion polypeptides
associated with hyperproliferative disorders, as well as nucleic acids
encoding the same and methods for detecting and identifying the same, are
provided. A feature of the subject fusion polypeptides is that they
include a C-terminal tyrosine kinase domain fused to an N-terminal domain
that is not normally fused to the C-terminal tyrosine kinase domain,
where the subject fusion polypeptides possess constitutively activated
tyrosine kinase activity. The subject fusion polypeptides are further
characterized in that they include at least one of the following
features: (a) the C-terminal domain is from a chromosome 4 tyrosine
kinase; (b) the N-terminal domain is from a chromosome 4 encoded protein,
e.g., a NM--030917 domain; and (c) the fusion protein does not arise
from a translocation event, i.e., it does not arise from the exchange of
DNA between different chromosomes; where in certain embodiments, at least
two of, including all three of, these features are present in the subject
fusion polypeptides. Also provided are methods of identifying and
characterizing the subject fusion proteins. Also provided are methods of
diagnosing disease conditions by detecting the presence of the subject
polypeptides/polynucleotides and/or detecting the deletions of one or
more genomic sequences, where the deletions result from the chromosomal
deletion event that gives rise to the subject
polypeptides/polynucleotides. In addition, screening assays for
identifying agents that find use in treating disease conditions
associated with the presence of the subject fusion polypeptides are
provided. Furthermore, methods of treating disease conditions with the
presence of the subject fusion polypeptides are provided.

[0013]Before the subject invention is described further, it is to be
understood that the invention is not limited to the particular
embodiments of the invention described below, as variations of the
particular embodiments may be made and still fall within the scope of the
appended claims. It is also to be understood that the terminology
employed is for the purpose of describing particular embodiments, and is
not intended to be limiting. Instead, the scope of the present invention
will be established by the appended claims.

[0014]In this specification and the appended claims, the singular forms
"a," "an" and "the" include plural reference unless the context clearly
dictates otherwise. Unless defined otherwise, all technical and
scientific terms used herein have the same meaning as commonly understood
to one of ordinary skill in the art to which this invention belongs.
Although any methods, devices and materials similar or equivalent to
those described herein can be used in the practice or testing of the
invention, the preferred methods, devices and materials are now
described.

[0015]Where a range of values is provided, it is understood that each
intervening value, to the tenth of the unit of the lower limit unless the
context clearly dictates otherwise, between the upper and lower limit of
that range, and any other stated or intervening value in that stated
range, is encompassed within the invention. The upper and lower limits of
these smaller ranges may independently be included in the smaller ranges,
and are also encompassed within the invention, subject to any
specifically excluded limit in the stated range. Where the stated range
includes one or both of the limits, ranges excluding either or both of
those included limits are also included in the invention.

[0016]All publications mentioned herein are incorporated herein by
reference for the purpose of describing and disclosing the elements that
are described in the publications which might be used in connection with
the presently described invention.

[0017]In further describing the subject invention, the subject oncokinase
fusion polypeptide and nucleic acid compositions are described first in
greater detail, followed by a more detailed review of the subject
antibody, diagnostic, screening and therapeutic embodiments of the
subject invention.

[0019]A feature of many embodiments of the subject fusion proteins is that
they confer an immortalized, and often hyperproliferative, phenotype onto
a cell in which they are present. In other words, cells that express the
subject fusion proteins are ones that have an immortalized and often
hyperproliferative phenotype. By "immortalized" is meant that the cell is
immortal as determined using the assay described in Lab. Invest. (2002)
82:323-333. By "hyperproliferative" is meant that the cell divides at an
above normal rate, as determined using the assay described in Cancer
Cell. (2002) 1: 421-432.

[0020]The subject fusion proteins are characterized by having a C-terminal
tyrosine kinase domain which is fused, either directly or through a
linking domain, to an N-terminal domain that is from a different protein,
i.e., is not from the same protein as the protein from which the
C-terminal tyrosine kinase is obtained. In certain embodiments, the
fusion of the N-terminal domain to the C-terminal tyrosine kinase domain
leads to or provides for the kinase domain being constitutively active,
as described above.

[0021]A further characteristic of the subject fusion polypeptides is that
they also include at least one of the following features:

[0022](1) they have a C-terminal chromosome 4 tyrosine kinase domain
(i.e., a chromosome 4 encoded tyrosine kinase encoded by a coding
sequence found on chromosome 4);

[0023](2) they have an N-terminal domain of a chromosome 4 protein (i.e.,
a chromosome 4 encoded protein encoded by a coding sequence found on
chromosome 4), e.g., NM--030917; and

[0024](3) they do not arise from a translocation event involving exchange
of genetic information between different chromosomes.

In certain embodiments, the fusion polypeptides include at least two of
the above features, and in certain of these embodiments the fusion
peptides include all three of the above features, e.g., both the C- and
N-terminal domains are from chromosome 4 encoded proteins.

[0025]By chromosome 4 tyrosine kinase is meant a tyrosine kinase whose
genomic coding sequence is located on the human chromosome 4. The
chromosome 4 tyrosine kinase domain of the subject fusion proteins may
include a domain or portion of a number of different chromosome 4
tyrosine kinases, where representative chromosome 4 tyrosine kinases of
interest include: PDGFRα, c-Kit and VEGFR-2. In many embodiments,
the chromosome 4 tyrosine kinase is PDGFRα.

[0026]The fusion polypeptides of the subject invention typically include
only a portion of the chromosome 4 tyrosine kinase, such that they do not
include the entire coding sequence for the chromosome 4 tyrosine kinase.
The portion is typically a C-terminal portion or domain of the chromosome
4 tyrosine kinase, which portion or domain exhibits kinase activity. The
length of the portions or domains present in the subject fusion
polypeptides is typically at least about 30% smaller, usually at least
about 40% smaller and more usually at least about 50% smaller (in terms
of residue number) than the full-length chromosome 4 tyrosine kinase of
which it is a portion. In many embodiments, the length of the C-terminal
chromosome 4 tyrosine kinase domains found in the subject fusion
polypeptides is at least about 400 residues, usually at least about 450
residues and more usually at least about 500 residues, where the length
of this C-terminal domain typically ranges in many embodiments from about
400 to about 1500, usually from about 500 to about 1200 and more usually
from about 500 to about 1000 residues.

[0027]As summarized above, the fusion polypeptides of the present
invention include an N-terminal domain of a protein that, when present in
the subject fusion proteins, results in the C-terminal kinase domain
being constitutively active. Accordingly, the N-terminal domain may be
considered to be a kinase activating protein. The N-terminal domain is,
in many embodiments, a domain of a chromosome 4 protein, where the
chromosome 4 protein is typically chromosomally located within proximity
to the chromosome 4 tyrosine kinase from which the C-terminal of the
fusion protein is derived. Since the N-terminal chromosome 4 protein is
located in proximity, the distance separating its genomic coding sequence
from that of the genomic coding sequence of the tyrosine kinase typically
does not exceed about 10 million base pairs, usually does not exceed
about 5 million base pairs and more usually does not exceed about 3
million base pairs.

[0028]In certain embodiments, the N-terminal domain of the subject fusion
polypeptides is a domain or portion of the chromosome 4 protein which is
encoded by the gene with the Genbank accession no. NM--030917, i.e.,
a "NM--030917 protein". In some prior publications, the
NM--030917 gene was identified by the Genbank accession no.
BC017724. This gene has recently been named Fip1L1 (see Cools et al., N.
Eng. J. Med. (2003) 348:1201-1214; and has also been named Rhe (See
Griffin et al., Proc. Nat'l Acad. Sci. USA (2003) 100: 7830-7835.

[0029]The fusion polypeptides of the subject invention typically include
only a portion of the chromosome 4 kinase activating protein, such that
they do not include the entire amino acid sequence of the protein. The
portion is typically an N-terminal portion or domain of the protein,
which, when fused to a C-terminal kinase domain in the fusion protein,
leads to constitutive activation of the kinase domain of the fusion
protein. The length of the portions or domains present in the subject
fusion polypeptides is typically at least about 1% smaller, usually at
least about 5% smaller and more usually at least about 10% smaller (in
terms of residue number) than the full-length chromosome 4 protein of
which it is a portion. In many embodiments, the length of the N-terminal
chromosome 4 kinase activating domains found in the subject fusion
polypeptides is at least about 50 residues, usually at least about 100
residues and more usually at least about 200 residues, where the length
of this N-terminal domain typically ranges in many embodiments from about
50 to about 350, usually from about 200 to about 350 and more usually
from about 300 to about 350 residues.

[0030]The subject fusion proteins range in length from about 500 to about
2000 residues, usually from about 700 to about 1500 residues and more
usually from about 800 to about 1200 amino acid residues, and the
projected molecular weight of the subject proteins based solely on the
number of amino acid residues in the protein ranges from about 55 to
about 220, usually from about 77 to about 165 and more usually from about
88 to about 132 kDa. As the subject fusion proteins may be modified,
e.g., phosphorylated, or modified in alternative ways, the actual
molecular weight of these proteins may be substantially higher than the
above projected molecular weights, typically ranging from about 1.1 to
about 2.0 times higher than the projected molecular weight.

[0031]Of particular interest in certain embodiment are fusion proteins
that have an amino acid sequence that is substantially the same as, or
identical to, the sequence appearing as SEQ ID NOs: 01, 02, 03 or 04, as
provided below.

[0032]By "substantially the same as" is meant a protein having a sequence
that has at least about 50%, usually at least about 60% and more usually
at least about 75%, and in many embodiments at least about 80%, usually
at least about 90% and more usually at least about 95%, 96%, 97%, 98% or
99% sequence identity with the sequence of the above provided sequences,
as measured by the BLAST compare two sequences program available on the
NCBI website using default settings, as measured over the entire length
of the protein, where the website has the address made up by placing
"www." in front of and ".gov" in back of "ncbi.nlm.nih".

[0033]In addition to the specific fusion proteins described above,
homologs or proteins (or fragments thereof) from other species, i.e.,
other animal species, are also provided, where such homologs or proteins
may be from a variety of different types of species, usually mammals,
e.g., rodents, such as mice, rats; domestic animals, e.g. horse, cow,
dog, cat; and primates, e.g., monkeys, baboons, humans etc. By homolog is
meant a protein having at least about 35%, usually at least about 40% and
more usually at least about 60% amino acid sequence identity to the
specific human fusion protein as identified above, where sequence
identity is determined using the algorithm described supra.

[0034]In certain embodiments, the fusion proteins of the subject invention
are present in a non-naturally occurring environment, e.g., are separated
from their naturally occurring environment. In certain embodiments, the
subject proteins are present in a composition that is enriched for the
subject proteins as compared to the subject proteins in their naturally
occurring environment. As such, purified fusion proteins according to the
subject invention are provided, where by purified is meant that the
proteins are present in a composition that is substantially free of
non-fusion proteins of the subject invention, where by substantially free
is meant that less than 90%, usually less than 60% and more usually less
than 50% of the composition is made up of non-fusion proteins of the
subject invention.

[0035]In certain embodiments of interest, the fusion proteins are present
in a composition that is substantially free of the constituents that are
present in its naturally occurring environment. For example, a human
fusion protein comprising a composition according to the subject
invention in this embodiment will be substantially, if not completely,
free of those other biological constituents, such as proteins,
carbohydrates, lipids, etc., with which it is present in its natural
environment. As such, protein compositions of these embodiments will
necessarily differ from those that are prepared by purifying the protein
from a naturally occurring source, where at least trace amounts of the
constituents or other components of the protein's naturally occurring
source will still be present in the composition prepared from the
naturally occurring source.

[0036]The fusion proteins of the subject invention may also be present as
isolates, by which is meant that the proteins are substantially free of
both non-fusion proteins and other naturally occurring biologic
molecules, such as oligosaccharides, polynucleotides and fragments
thereof, and the like, where substantially free in this instance means
that less than 70%, usually less than 60% and more usually less than 50%
(by dry weight) of the composition containing the isolated fusion
proteins is a non-fusion protein naturally occurring biological molecule.
In certain embodiments, the fusion proteins are present in substantially
pure form, where by substantially pure form is meant at least 95%,
usually at least 97% and more usually at least 99% pure.

[0037]In addition to the naturally occurring proteins, polypeptides that
vary from the naturally occurring proteins are also provided. By
polypeptide is meant proteins having an amino acid sequence encoded by an
open reading frame (ORF) of a fusion protein coding sequence, described
below, including the full length protein and fragments thereof,
particularly biologically active fragments and/or fragments corresponding
to functional domains, and including fusions of the subject fusion
proteins to yet additional proteins or parts thereof, e.g.,
immunoglobulin domains, peptididic tags; and the like. Fragments of
interest will typically be at least about 10 aa in length, usually at
least about 50 aa in length, and may be as long as 300 aa in length or
longer, but will usually not exceed about 1000 aa in length.

Nucleic Acid Compositions

[0038]Also provided are nucleic acid compositions that encode the subject
fusion polypeptides and fragments thereof, etc., as described above.
Specifically, nucleic acid compositions encoding the subject
polypeptides, as well as fragments or homologs thereof, are provided. By
"nucleic acid composition" is meant a composition comprising a sequence
of nucleotide bases that encodes a fusion polypeptide to the subject
invention, i.e., a region of genomic DNA capable of being transcribed
into mRNA that encodes a fusion polypeptide of the subject invention, the
mRNA that encodes and directs the synthesis of a fusion polypeptide of
the subject invention, the cDNA derived from reverse transcription of the
mRNA, etc. Specific nucleic acids of interest include those identified
herein as SEQ ID NO:05; SEQ ID NO:06; SEQ ID NO:07 and SEQ ID NO:08. Also
encompassed in this term are nucleic acids that are homologous,
substantially similar or identical to the nucleic acids specifically
disclosed herein, e.g., SEQ ID NO:05; SEQ ID NO:06; SEQ ID NO:07 and SEQ
ID NO:08, where sequence similarity is determined using the BLAST compare
functionality provided online by the National Center for Biotechnology
(using default settings).

[0039]Also provided are nucleic acids that are homologous to the provided
nucleic acids, at least with respect to the coding regions thereof. The
source of homologous nucleic acids to those specifically listed above may
be any mammalian species, e.g., primate species, particularly human;
rodents, such as rats and mice, canines, felines, bovines, equines, etc;
as well as non-mammalian species, e.g., yeast, nematodes, etc. Between
mammalian species, e.g., human and mouse, homologs typically have
substantial sequence similarity, e.g., at least 75% sequence identity,
usually at least 90%, more usually at least 95% between nucleotide
sequences. Sequence similarity is calculated based on a reference
sequence, which may be a subset of a larger sequence, such as a conserved
motif, coding region, flanking region, etc. A reference sequence will
usually be at least about 18 nt long, more usually at least about 30 nt
long, and may extend to the complete sequence that is being compared.
Algorithms for sequence analysis are known in the art, such as BLAST,
described in Altschul et al. (1990), J. Mol. Biol. 215:403-10 (using
default settings, i.e. parameters w=4 and T=17). Unless indicated
otherwise, the sequence similarity values reported herein are those
determined using the above referenced BLAST program using default
settings. Of particular interest in certain embodiments are nucleic acids
including a sequence substantially similar to the specific nucleic acids
identified above, where by substantially similar is meant having sequence
identity to this sequence of at least about 90%, usually at least about
95% and more usually at least about 99%.

[0040]Also provided are nucleic acids that hybridize to the
above-described nucleic acids under stringent conditions. An example of
stringent hybridization conditions is overnight incubation 42° C.
a 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50
mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextran
sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by
washing the filters in 0.1×SSC at about 65° C. Stringent
hybridization conditions are hybridization conditions that are at least
as stringent as the above representative conditions. Other stringent
hybridization conditions are known in the art and may also be employed to
identify nucleic acids of this particular embodiment of the invention.

[0041]Nucleic acids encoding the proteins and polypeptides of the subject
invention may be cDNAs or genomic DNAs, as well as fragments thereof. The
nucleic acids may also be mRNAs, e.g., transcribed from genomic DNA, that
encode (i.e. are translated into) the subject proteins and polypeptides.
Also provided are genes encoding the subject proteins, where the term
"gene" means the open reading frame encoding specific proteins and
polypeptides, and introns that are present in the open reading frame, as
well as adjacent 5' and 3' non-coding nucleotide sequences involved,
e.g., untranslated regions, promoter or other regulatory elements, etc.,
in the regulation of expression, up to about 20 kb beyond the coding
region, but possibly further in either direction. The gene may be
introduced into an appropriate vector for extrachromosomal maintenance or
for integration into a host genome.

[0042]The term "cDNA" as used herein is intended to include all nucleic
acids that share the arrangement of sequence elements found in native
mature mRNA species or the complementary sequences thereof, where
sequence elements at least include exons. Normally mRNA species have
contiguous exons, with the intervening introns, when present, being
removed by nuclear RNA splicing, to create a continuous open reading
frame encoding an oncokinase fusion protein according to the subject
invention.

[0043]A genomic sequence of interest comprises the nucleic acid present
between the initiation codon and the stop codon, as defined in the listed
sequences, including all of the introns that are normally present in a
native chromosome. It may further include specific transcriptional and
translational regulatory sequences, such as promoters, enhancers, etc.,
including about 1 kb, but possibly more, of flanking genomic DNA at
either the 5' or 3' end of the transcribed region. The genomic DNA may be
isolated as a fragment of 100 kbp or smaller; and substantially free of
flanking chromosomal sequence. The genomic DNA flanking the coding
region, either 3' or 5', or internal regulatory sequences as sometimes
found in introns, contains sequences required for proper tissue and stage
specific expression.

[0044]The nucleic acid compositions of the subject invention may encode
all or a part of the subject proteins and polypeptides, described in
greater detail above. Double or single stranded fragments may be obtained
from the DNA sequence by chemically synthesizing oligonucleotides in
accordance with conventional methods, by restriction enzyme digestion, by
PCR amplification, etc. For the most part, DNA fragments will be of at
least 15 nt, usually at least 18 nt or 25 nt, and may be at least about
50 nt.

[0045]The nucleic acids of the subject invention are isolated and obtained
in substantial purity, generally as other than an intact chromosome.
Usually, the DNA will be obtained substantially free of other nucleic
acid sequences that do not include a fusion protein sequence or fragment
thereof, generally being at least about 50%, usually at least about 90%
pure and are typically "recombinant," i.e. flanked by one or more
nucleotides with which it is not normally associated on a naturally
occurring chromosome.

[0046]In addition to the plurality of uses described in greater detail in
following sections, the subject nucleic acid compositions find use in the
preparation of all or a portion of the subject polypeptides, as described
above.

[0047]Also provided are nucleic acid probes, as well as constructs, e.g.,
vectors, expression systems, etc., as described more fully below, that
include a nucleic acid sequence as described above. Probes of the subject
invention are generally fragments of the provided nucleic acid. The
probes may be a large or small fragment, generally ranging in length from
about 10 to 100 nt, usually from about 15 to 50 nt. In using the subject
probes, nucleic acids having sequence similarity are detected by
hybridization under low stringency conditions, for example, at 50°
C. and 6×SSC (0.9 M sodium chloride/0.09 M sodium citrate)(or
analogous conditions) and remain bound when subjected to washing at
higher stringency conditions, e.g., 55° C. in 1×SSC (0.15 M
sodium chloride/0.015 M sodium citrate) (or analogous conditions).
Sequence identity may be determined by hybridization under stringent
conditions, for example, at 50° C. or higher and 0.1×SSC (15
mM sodium chloride/01.5 mM sodium citrate)(or analogous conditions).
Nucleic acids having a region of substantial identity to the provided
nucleic acid sequences bind to the provided sequences under stringent
hybridization conditions. By using probes, particularly labeled probes of
DNA sequences, one can isolate homologous or related sequences.

[0048]The subject nucleic acids may be produced using any convenient
protocol, including synthetic protocols, e.g., such as those where the
nucleic acid is synthesized by a sequential monomeric approach (e.g., via
phosphoramidite chemistry); where subparts of the nucleic acid are so
synthesized and then assembled or concatamerized into the final nucleic
acid, and the like. Where the nucleic acid of interest has a sequence
that occurs in nature, the nucleic acid may be retrieved, isolated,
amplified etc., from a natural source using conventional molecular
biology protocols.

[0049]Also provided are constructs comprising the subject nucleic acid
compositions, e.g., those that include a fusion protein coding sequence,
inserted into a vector, where such constructs may be used for a number of
different applications, including propagation, screening, genome
alteration, and the like, as described in greater detail below.
Constructs made up of viral and non-viral vector sequences may be
prepared and used, including plasmids, as desired. The choice of vector
will depend on the particular application in which the nucleic acid is to
be employed. Certain vectors are useful for amplifying and making large
amounts of the desired DNA sequence. Other vectors are suitable for
expression in cells in culture, e.g., for use in screening assays. Still
other vectors are suitable for transfer and expression in cells in a
whole animal, e.g., in the production of animal models of
hyperproliferative diseases. The choice of appropriate vector is well
within the ability of those of ordinary skill in the art. Many such
vectors are available commercially. To prepare the constructs, the
partial or full-length nucleic acid is inserted into a vector typically
by means of DNA ligase attachment to a cleaved restriction enzyme site in
the vector. Alternatively, the desired nucleotide sequence can be
inserted by homologous recombination in vivo. Typically, homologous
recombination is accomplished by attaching regions of homology to the
vector on the flanks of the desired nucleotide sequence. Regions of
homology are added by ligation of oligonucleotides, or by polymerase
chain reaction using primers that include both the region of homology and
a portion of the desired nucleotide sequence, for example. Yet another
means to insert the nucleic acids into appropriate vectors is to employ
one of the increasingly employed recombinase based methods for
transferring nucleic acids among vectors, e.g., the Creator® system
from Clontech; the Gateway® system from Invitrogen, etc.

[0050]Also provided are expression cassettes that include a coding
sequence. By expression cassette is meant a nucleic acid that includes a
sequence encoding a subject peptide or protein operably linked to a
promoter sequence, where by operably linked is meant that expression of
the coding sequence is under the control of the promoter sequence.

Preparation of Polypeptides According to the Subject Invention

[0051]The subject fusion proteins may be obtained using any convenient
protocol. As such, they may be obtained from naturally occurring sources
or recombinantly produced. Naturally occurring sources of the subject
proteins include tissues and portions/fractions, including cells, cell
lines and fractions thereof, e.g., extracts, homogenates etc., that
include cells in which the desired protein is expressed.

[0052]The subject proteins may also be obtained from synthetic protocols,
e.g., by expressing a recombinant gene encoding the subject protein, such
as the polynucleotide compositions described above, in a suitable host
under conditions sufficient for post-translational modification to occur
in a manner that provides the expressed fusion protein with the desired
constitutively active kinase activity. For expression, an expression
cassette may be employed. The expression cassette or vector will provide
a transcriptional and translational initiation region, which may be
inducible or constitutive, where the coding region is operably linked
under the transcriptional control of the transcriptional initiation
region, and under the translational control of the translational
initiation region, and a transcriptional and translational termination
region. These control regions may be native to a gene of the subject
invention, or may be derived from exogenous sources.

[0053]Expression cassettes may be prepared comprising a transcription
initiation region, the nucleic acid coding sequence or fragment thereof,
and a transcriptional termination region. Of particular interest is the
use of sequences that allow for the expression of functional epitopes or
domains, usually at least about 8 amino acids in length, more usually at
least about 15 amino acids in length, to about 25 amino acids, and up to
the complete open reading frame of the coding sequence. After
introduction of the DNA, the cells containing the construct may be
selected by means of a selectable marker, the cells expanded and then
used for expression.

[0054]The subject proteins and polypeptides may be expressed in
prokaryotes or eukaryotes in accordance with conventional ways, depending
upon the purpose for expression. For large scale production of the
protein, a unicellular organism, such as E. coli, B. subtilis, S.
cerevisiae, insect cells in combination with baculovirus vectors, or
cells of a higher organism such as vertebrates, particularly mammals,
e.g. COS 7 cells, may be used as the expression host cells. In some
situations, it is desirable to express the gene in eukaryotic cells,
where the encoded protein will benefit from native folding and
post-translational modifications. Small peptides can also be synthesized
in the laboratory. Polypeptides that are subsets of the complete sequence
may be used to identify and investigate parts of the protein important
for function.

[0055]Specific expression systems of interest include bacterial, yeast,
insect cell and mammalian cell derived expression systems. Representative
systems from each of these categories are provided below:

[0064]When any of the above host cells, or other appropriate host cells or
organisms, are used to replicate and/or express the polynucleotides or
nucleic acids of the invention, the resulting replicated nucleic acid,
RNA, expressed protein or polypeptide, is within the scope of the
invention as a product of the host cell or organism.

[0065]Once the source of the protein is identified and/or prepared, e.g.,
a transfected host expressing the protein is prepared, the protein is
then purified to produce the desired fusion protein comprising
composition. Any convenient protein purification procedures may be
employed, where suitable protein purification methodologies are described
in Guide to Protein Purification, (Deuthser ed.) (Academic Press, 1990).
For example, a lysate may be prepared from the original source, e.g.
naturally occurring cells or tissues that express the subject fusion
proteins or the expression host expressing the subject fusion proteins,
and purified using HPLC, exclusion chromatography, gel electrophoresis,
affinity chromatography, and the like.

[0066]Also provided by the subject invention are methods for identifying
and characterizing onto-tyrosine kinase fusion proteins in a sample,
e.g., a cell, tissue or other sample of interest. In such methods,
onco-tyrosine kinase fusion proteins are identified by first screening
the sample of interest to determine whether or not any onco-tyrosine
kinase fusion proteins are present in the sample. To screen a sample,
tyrosine-phosphorylated proteins are typically first separated from the
remaining constituents of said sample to produce a population of sample
derived tyrosine phosphorylated proteins. Separation or isolation of the
tyrosine-kinase fusion proteins from the remaining components in the same
can be accomplished using any convenient protocol, e.g., via
immunoprecipitation with an anti-phosphotyrosine antibody. Next, the
constituent members of the obtained population of sample derived tyrosine
phosphorylated proteins are evaluated for the presence of domains from
two or more different proteins. In other words, one or more of the
different proteins in the isolated population of tyrosine phosphorylated
proteins are evaluated to determine whether they include domains from two
or more different proteins. This evaluating step may be accomplished
using any convenient protocol. In one representative embodiment, the
population of tyrosine phosphorylated proteins is separated or
fractionated into its constituent proteins using, for example, SDS-PAGE,
2-dimensional IE/PAGE, high-performance liquid chromatography, capillary
electrophoresis, etc. Next, the constituent proteins are cleaved into
smaller sized peptides, e.g., via subjection to proteolysis using an
endoproteinase such as trypsin. The resultant peptides are then separated
or fractionated using, for example, microbore capillary electrophoresis,
high-performance liquid chromatography, or mass spectrometry. The
resultant isolated or fractionated peptides are then sequenced using, for
example, automated Edman degradation or mass spectrometry. The resultant
sequences are then compared to the sequences of the observed peptides
with known or predicted peptide sequences from proteins expressed in the
organism from which the sample was obtained. Next, a determination is
made as to which of the constituent proteins provides proteolysis
peptides from two different proteins, for example, peptides from the
N-terminal domain of the protein encoded by gene NM--030917 and
peptides from the C-terminal domain of PDGFRα. In this manner, a
sample of interest is screened for onco-tyrosine kinase fusion proteins.

[0067]Any identified onco-tyrosine kinase fusion proteins can be further
characterized to identify one or more of: (a) the full amino acid
sequence; (b) the sequence of an encoding nucleic acid, e.g., mRNA
encoding the fusion protein; (c) the sequence of the gene or genomic DNA
encoding the fusion protein, etc. For example, the observed peptide
sequences of the identified fusion protein can be employed to design PCR
primers that allow for amplification of all or part of an mRNA encoding
the fusion protein, for example, the region of the mRNA that comprises
the fusion junction. The resultant amplified cDNA encoding all or part of
the fusion protein can then be directly sequenced or first cloned and the
sequence of the cloned cDNA encoding all or part of the fusion protein
determined using, for example, automated dideoxy DNA sequencing. The
resultant sequence can then be used to determine the cDNA sequence to
predict the sequence of a novel proteolytic peptide encompassing the
fusion junction within the fusion protein. The presence of the novel
proteolytic peptide encompassing the fusion junction can then be
determined by for example, mass spectrometry or automated Edman
degradation. The determined cDNA sequence can also be employed to design
primers for amplification of genomic DNA encompassing the fusion
junction. The resultant amplified genomic DNA sequence encompassing the
fusion junction can then be directly sequenced or first cloned and the
sequence of the cloned genomic DNA determined using, for example,
automated dideoxy DNA sequencing. In this way, the identified fusion
proteins are further characterized.

Antibodies

[0068]Also provided are antibodies that bind to the subject fusion
proteins and homologs thereof. Suitable antibodies are obtained by
immunizing a host animal with peptides comprising all or a portion of the
fusion protein. Suitable host animals include rat, sheep, goat, hamster,
rabbit, etc. The origin of the protein immunogen may be mouse, rat,
monkey etc, but is human in many embodiments. The host animal will
generally be a different species than the immunogen, e.g. human protein
used to immunize rabbit, etc.

[0069]The immunogen may include the complete protein, or fragments and
derivatives thereof; e.g., a fragment comprising the unique sequence
found at the site of fusion of the N- and C-terminal domains. Immunogens
employed in certain embodiments include all or a part of the subject
fusion protein, where these residues contain any post-translation
modifications, such as glycosylation, found on the native target protein.
Immunogens comprising the fusion protein are produced in a variety of
ways known in the art, e.g. expression of cloned genes using conventional
recombinant methods, isolation from HEC, etc.

[0070]For preparation of polyclonal antibodies, the first step is
immunization of the host animal with the target protein, where the target
protein will preferably be in substantially pure form, comprising less
than about 1% contaminant. The immunogen may include the complete target
protein, fragments or derivatives thereof. To increase the immune
response of the host animal, the target protein may be combined with an
adjuvant, where suitable adjuvants include alum, dextran, sulfate, large
polymeric anions, oil and water emulsions, e.g. Freund's adjuvant,
Freund's complete adjuvant, and the like. The target protein may also be
conjugated to synthetic carrier proteins or synthetic antigens. A variety
of hosts may be immunized to produce the polyclonal antibodies. Such
hosts include rabbits, guinea pigs, mice, rats, sheep, goats, and the
like. The target protein is administered to the host, usually
intradermally, with an initial dosage followed by one or more, usually at
least two, additional booster dosages. Following immunization, the blood
from the host will be collected, followed by separation of the serum from
the blood cells. The Ig present in the resultant antiserum may be further
fractionated using known methods, such as ammonium salt fractionation,
DEAE chromatography, and the like.

[0071]Monoclonal antibodies of the subject invention may be produced by
conventional techniques. Generally, the spleen and/or lymph nodes of an
immunized host animal provide a source of plasma cells. The plasma cells
are immortalized by fusion with myeloma cells to produce hybridoma cells.
Culture supernatant from individual hybridomas is screened using standard
techniques to identify those producing antibodies with the desired
specificity. Suitable animals for production of monoclonal antibodies to
the human protein include mouse, rat, hamster, etc. To raise antibodies
against the human protein, the animal will generally be a hamster, guinea
pig, rabbit, etc. The antibody may be purified from the hybridoma cell
supernatants or ascites fluid by conventional techniques, e.g. affinity
chromatography using MPTS bound to an insoluble support, protein A
sepharose, etc.

[0072]The antibody may be produced as a single chain, instead of the
normal multimeric structure. Single chain antibodies are described in
Jost et al. (1994) J. Biol. Chem. 269:26267-73, and others. DNA sequences
encoding the variable region of the heavy chain and the variable region
of the light chain are ligated to a spacer encoding at least about 4
amino acids of small neutral amino acids, including glycine and/or
serine. The protein encoded by this fusion allows assembly of a
functional variable region that retains the specificity and affinity of
the original antibody.

Diagnostic Applications

[0073]Also provided are methods of diagnosing disease states associated
with fusion protein activity (or even the absence thereof)(such as those
disease conditions listed below), e.g., based on detecting/observing
levels of fusion protein or the presence and/or expression level of the
gene/coding sequence in a biological sample of interest, and/or detecting
the deletions of one or more nucleic acid (particularly genomic)
sequences in a sample of interest, where the deletions result from the
chromosomal deletion event that gives rise to the subject
polypeptides/polynucleotides.

[0074]Samples, as used herein, include biological fluids such as blood,
cerebrospinal fluid, tears, saliva, lymph, dialysis fluid and the like;
organ or tissue culture derived fluids; and fluids extracted from
physiological tissues. Also included in the term are derivatives and
fractions of such fluids. Samples may also include cells, which may be
solitary or in need of being dissociated in the case of solid tissues.
Alternatively tissue sections may be analyzed or a lysate of the cells
may be prepared.

[0075]A number of methods are available for determining the presence
and/or expression level of a gene or protein in a particular sample. For
example, diagnosis may be performed by a number of methods to determine
the absence or presence or altered amounts of fusion protein in a patient
sample. For example, detection may utilize staining of cells or
histological sections with labeled antibodies, performed in accordance
with conventional methods. Cells are permeabilized to stain intracellular
molecules. The antibodies of interest are added to the cell sample, and
incubated for a period of time sufficient to allow binding to the
epitope, usually at least about 10 minutes. The antibody may be labeled
with radioisotopes, enzymes, fluorophores, chemiluminescers, or other
labels for direct detection. Alternatively, a second stage antibody or
reagent is used to amplify the signal. Such reagents are well known in
the art. For example, the primary antibody may be conjugated to biotin,
with horseradish peroxidase-conjugated avidin added as a second stage
reagent. Final detection uses a substrate that undergoes a color change
in the presence of the peroxidase. Alternatively, the secondary antibody
conjugated to a flourescent compound, e.g. fluorescein, rhodamine, Texas
red, etc. The absence or presence of antibody binding may be determined
by various methods, including flow cytometry of dissociated cells,
microscopy, radiography, scintillation counting, etc.

[0076]Alternatively, one may focus on the presence of a gene encoding the
fusion protein and/or expression of the fusion protein. A number of
methods are available for analyzing nucleic acids for the presence of a
specific sequence, e.g., a coding sequence for the subject fusion
proteins. Where large amounts of DNA are available, genomic DNA is used
directly. Alternatively, the region of interest is cloned into a suitable
vector and grown in sufficient quantity for analysis. Cells that express
the fusion protein may be used as a source of mRNA, which may be assayed
directly or reverse transcribed into cDNA for analysis. The nucleic acid
may be amplified by conventional techniques, such as the polymerase chain
reaction (PCR), to provide sufficient amounts for analysis. The use of
the polymerase chain reaction is described in Saiki, et al. (1985),
Science 239:487, and a review of techniques may be found in Sambrook, et
al. Molecular Cloning: A Laboratory Manual, CSH Press 1989, pp.
14.2-14.33. Alternatively, various methods are known in the art that
utilize oligonucleotide ligation as a means of detecting polymorphisms,
for examples see Riley et al. (1990), Nucl. Acids Res. 18:2887-2890; and
Delahunty et al. (1996), Am. J. Hum. Genet. 58:1239-1246.

[0077]A detectable label may be included in an amplification reaction.
Suitable labels include fluorochromes, e.g. fluorescein isothiocyanate
(FITC), rhodamine, Texas Red, phycoerythrin, allophycocyanin,
6-carboxyfluorescein (6-FAM),
2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE),
6-carboxy-X-rhodamine (ROX), 6-carboxy-2',4',7',4,7-hexachlorofluorescein
(HEX), 5-carboxyfluorescein (5-FAM) or
N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), radioactive labels,
e.g. 32P, 35S, 3H; etc. The label may be a two-stage
system, where the amplified DNA is conjugated to biotin, haptens, etc.
having a high affinity binding partner, e.g. avidin, specific antibodies,
etc., where the binding partner is conjugated to a detectable label. The
label may be conjugated to one or both of the primers. Alternatively, the
pool of nucleotides used in the amplification is labeled, so as to
incorporate the label into the amplification product.

[0078]The sample nucleic acid, e.g., amplified or cloned fragment, may be
analyzed for variations from the specifically provided "wild type"
sequences provided herein by one of a number of methods known in the art.
The nucleic acid may be sequenced by dideoxy or other methods, and the
sequence of bases compared to a wild-type sequence. Hybridization with
the variant sequence may also be used to determine its presence, by
Southern blots, dot blots, etc. The hybridization pattern of a control
and variant sequence to an array of oligonucleotide probes immobilized on
a solid support, as described in U.S. Pat. No. 5,445,934, or in WO
95/35505, may also be used as a means of detecting the presence of
variant sequences. Single strand conformational polymorphism (SSCP)
analysis, denaturing gradient gel electrophoresis (DGGE), and
heteroduplex analysis in gel matrices are used to detect conformational
changes created by DNA sequence variation as alterations in
electrophoretic mobility. Alternatively, where a polymorphism creates or
destroys a recognition site for a restriction endonuclease, the sample is
digested with that endonuclease, and the products size fractionated to
determine whether the fragment was digested. Fractionation is performed
by gel or capillary electrophoresis, particularly acrylamide or agarose
gels.

[0079]Screening for variants or mutations may be based on the functional
or antigenic characteristics of the protein. Protein truncation assays
are useful in detecting deletions that may affect the biological activity
of the protein. Various immunoassays designed to detect polymorphisms in
proteins may be used in screening. Where many diverse genetic mutations
lead to a particular disease phenotype, functional protein assays have
proven to be effective screening tools. The activity, e.g. kinase
functionality, of the encoded protein may be determined by comparison
with the wild-type protein.

[0080]Diagnostic methods of the subject invention in which the level of
expression is of interest will typically involve comparison of the
nucleic acid abundance of a sample of interest with that of a control
value to determine any relative differences, where the difference may be
measured qualitatively and/or quantitatively, which differences are then
related to the presence or absence of an abnormal expression pattern. A
variety of different methods for determining the nucleic acid abundance
in a sample are known to those of skill in the art, where particular
methods of interest include those described in: Pietu et al., Genome Res.
(June 1996) δ: 492-503; Zhao et al., Gene (Apr. 24, 1995) 156:
207-213; Soares, Curr. Opin. Biotechnol. (October 1997) 8: 542-546;
Ravel, J. Pharmacol Toxicol Methods (November 1994) 32: 125-127;
Chalifour et al., Anal. Biochem (Feb. 1, 1994) 216: 299-304; Stolz &
Tuan, Mol. Biotechnol. (December 19960 6: 225-230; Hong et al.,
Bioscience Reports (1982) 2: 907; and McGraw, Anal. Biochem. (1984) 143:
298. Also of interest are the methods disclosed in WO 97/27317, the
disclosure of which is herein incorporated by reference.

[0081]In certain embodiments, the diagnostic applications may further
include a further fusion protein characterization step, where the
presence of the fusion protein of interest is found. For example, one may
further characterize the particular fusion point of the fusion protein,
where knowledge of the particular fusion point is of use, e.g., in
developing rational treatment protocols, as described below. One may also
further characterize the fusion protein to determine whether it is
resistant to any particular contemplated therapeutic agent. For example,
the fusion protein may include a mutation or variation as compared to the
"wild-type" sequence that confers resistance to a particular
pharmacological agent. A specific representative embodiment of such a
mutation or alteration is the T674I mutation in the
NM--030917-PDGFRα fusion protein, as described in greater
detail in the experimental section, below.

[0082]In certain embodiments, instead of (or in addition to) detecting the
presence of the subject fusion proteins and/or nucelci acids encoding the
same, as described above, the absence of one or more nucleic acids, e.g.,
genomic sequences, is detected, where the absence of the one or more
nucleic acids occurs because of a chromosomal deletion event that results
in the presence of the subject fusion proteins, and therefore can be
employed to determine the presence of the subject fusion proteins.

[0083]Specifically, since the subject fusion kinases of the present
invention result from a chromosomal deletion event, one can determine the
presence of the subject fusion kinases by screening or assaying for the
presence of one or more particular genetic loci that are located between
NM--030917 and PDGFRα and are not present if the chromosomal
deletion event has occurred. Representative genomic sequences that can be
assayed in these embodiments of the subject methods include sequences
found in genes that are located between NM--030917 and PDGFRα,
like the genes LNX, RPL21, CHIC2, MORF4 or GSH2. Alternatively, one can
also assay for sequences at 4q12 that do not code for a gene, where the
sequence is specific for 4q12. In yet other embodiments, one may screen
for sequences at the very 3' end of NM--030917 or the 5' end of
PDGFRα, including sequences coding for the extracellular domain of
PDGFRα but not of sequences coding for the transmembrane of
intracellular domain of PDGFRα.

[0084]In these embodiments, the presence or absence of the target
sequences, as described above, can be assayed using any convenient
protocol. For example, the detection of particular genetic loci can be
achieved by using fluorescence in situ hybridization (FISH). In this
technique, a fluorescently labeled oligonucleotide is used as a probe
that will hybridize with its complementary sequence on the respective
chromosome if that complementary sequence is present, which hybridization
can then be detected by fluorescent microscopy. In the present situation,
given that the 4q12 locus is present twice in the genetic material of
each cell, hybridization of a probe derived from that region should
result in two distinct hybridization events and, hence, in two
fluorescent signals per normal nucleus, if no chromosomal deletion has
occurred. Representative probes that can be employed include, but are not
limited to: (a) those derived from genes that are located between
NM--030917 and PDGFRα, such as the genes LNX, RPL21, CHIC2,
MORF4 or GSH2; (b) probes derived from sequences at 4q12 that do not code
for a gene; and (3) probes derived from a sequence at the very 3' end of
NM--030917 or the 5' end of PDGFRα, including sequences coding
for the extracellular domain of PDGFRα. If a deletion at the 4q12
locus has occurred, only one hybridization signal per nucleus will be
detected. The presence of only one hybridization signal per nucleus in a
significant number (e.g., at least about 100, such as about 200 or more)
of hematological cells derived from the bone marrow or from peripheral
blood of a patient is strongly indicative of a deletion that can have led
to the formation of the fusion gene and therefore can be employed as a
diagnostic marker.

Screening Assays

[0085]Also provided by the subject invention are screening protocols and
assays for identifying agents that modulate, e.g., inhibit or enhance,
activity of the subject fusion proteins. As such, the screening assays
are assays that provide for the identification of agents that modulate,
e.g., inhibit or enhance, the kinase activity of the subject fusion
proteins.

[0086]The screening methods will typically be assays that provide for
qualitative/quantitative measurements of fusion protein kinase activity,
e.g., of the ability of the fusion protein to catalyze transfer of a
phosphoryl group from a donor to an acceptor. For example, the assay
could be an assay which measures the kinase activity of a fusion protein
of the subject invention in the presence and absence of a candidate
inhibitor agent. The screening method may be an in vitro or in vivo
format, where both formats are readily developed by those of skill in the
art. In other words, such assays can be done in vivo or in vitro in
mammalian cells, non-mammalian cells, yeast, bacteria, etc.

[0087]A. In Vitro Models of Fusion Protein Function

[0088]In vitro models of fusion protein function are provided, where the
in vitro models may be cell-free models or employ the use of cells. Of
particular interest are models of fusion protein kinase activity.

[0089]Cell free-models typically include: a fusion protein polypeptide and
a candidate modulatory agent, e.g., competitor or inhibitor
agent/molecule, where the models further typically include at least one
of: a donor molecule that includes a phsophoryl group (typically ATP)
that is to be transferred to an acceptor molecule and an acceptor
molecule that is to receive the phosphoryl group transferred by the
donor.

[0090]The competitor my be any compound that is, or is suspected to be, a
compound capable of specifically inhibiting the fusion protein. Depending
on the particular model, one or more of, usually one of, the specified
components may be labeled, where by labeled is meant that the components
comprise a detectable moiety, e.g., a fluorescent or radioactive tag, or
a member of a signal producing system, e.g. biotin for binding to an
enzyme-streptavidin conjugate in which the enzyme is capable of
converting a substrate to a chromogenic product.

[0091]The above cell free in vitro models may be designed a number of
different ways, where a variety of assay configurations and protocols may
be employed, as are known in the art. For example, one of the components
may be bound to a solid support, and the remaining components contacted
with the support bound component. The above components of the method may
be combined at substantially the same time or at different times, e.g.
soluble fusion protein and a competitor ligand may be combined first, and
the resultant mixture subsequently combined with bound acceptor molecule.
Following the contact step, the subject methods will generally, though
not necessarily, further include a washing step to remove unbound
components, where such a washing step is generally employed when required
to remove label that would give rise to a background signal during
detection, such as radioactive or fluorescently labeled non-specifically
bound components. Following the optional washing step, the presence of
bound fusion protein will then be detected.

[0092]In alternative in vitro models, the above components may be present
in a cell free environment in which the fusion protein exhibits kinase
activity in the absence of any inhibitor. The kinase activity is then
monitored in the presence and absence of candidate modulating agents,
where kinase activity may be determined using any convenient assay, e.g.,
the kinase activity assay described in Proc. Nat'l Acad. Sci. USA (2002)
97:2419-2424.

[0093]Also of interest are in vitro models in which cells are employed.
There are numerous cell containing in vitro assays which can be readily
adapted by those of skill in the art for the purposes described herein.
For example, the activity of an inhibitor of the subject oncokinase
fusion polypeptides can be assessed biochemically using cells expressing
the oncogene. Those cells can be a cancer cell line or they can be a cell
line generated from the transfection of fusion kinase cDNA. As a
reflection of its constitutive activity, fusion kinase polypeptide
autophosphorylates. If cells are incubated with an inhibitor, the level
of kinase inhibition can be evaluated by immunoprecipitating the fusion
kinase polypeptide and subsequently, by performing a Western Blot
analysis in which the blotting antibody is selective for phosphotyrosine
residues. The level of kinase inhibition is reflected in the decrease of
tyrosine phosphorylation of the fusion kinase polypeptide.

[0094]In another embodiment, the inhibition of the fusion kinase
polypeptide with an inhibitor leads to cellular responses in the
respective primary cancer cells and cancer cell lines or appropriate cell
lines derived from the transfection of the fusion kinase polypeptide
cDNA. The cellular responses can include decreased proliferation,
differentiation or apoptosis. The inhibition of proliferation can be
assessed by a multitude of assays, including simple cell counting as well
as incorporation of BrdU into DNA followed by ELISA. Induction of
apoptosis can be evaluated by annexin staining using flow cytometry,
assays for the functional integrity of mitochondria (MTT assays) as well
as assays to monitor caspase activation or DNA fragmentation.

[0095]B. In Vivo Models of Fusion Protein Function

[0096]A variety of different in vivo models of fusion protein function are
also provided by the subject invention and may be used in the screening
assays of the subject invention. In vivo models of interest include
engineered cells that include an expression cassette encoding the subject
fusion proteins. Also of interest in the subject screening assays are
multicellular in vivo models, e.g., the transgenic animal models
described below.

[0097]The subject nucleic acids can be used to generate transgenic,
non-human animals or site-specific gene modifications in cell lines.
Transgenic animals may be made through homologous recombination, where
the normal locus is altered. Alternatively, a nucleic acid construct is
randomly integrated into the genome. Vectors for stable integration
include plasmids, retroviruses and other animal viruses, YACs, and the
like.

[0098]The modified cells or animals are useful in the study of fusion
protein function and regulation. Specific constructs of interest include
anti-sense, which will block expression, expression of dominant negative
mutations, and over-expression of fusion protein genes. Where a sequence
is introduced, the introduced sequence may be either a complete or
partial sequence of a gene that is exogenous to the host animal, e.g., a
human sequence. A detectable marker, such as lac Z, may be introduced
into the locus, where upregulation of expression will result in an easily
detected change in phenotype.

[0099]One may also provide for expression of the gene or variants thereof
in cells or tissues where it is not normally expressed (e.g., mammalian,
non-mammalian, yeast, bacterial, etc. cells), at levels not normally
present in such cells or tissues, or at abnormal times of development.
DNA constructs for homologous recombination will comprise at least a
portion of the gene native to the species of the host animal, wherein the
gene has the desired genetic modification(s), and includes regions of
homology to the target locus. DNA constructs for random integration need
not include regions of homology to mediate recombination. Conveniently,
markers for positive and negative selection are included. Methods for
generating cells having targeted gene modifications through homologous
recombination are known in the art. For various techniques for
transfecting mammalian cells, see Keown et al. (1990), Meth. Enzymol.
185:527-537.

[0100]For embryonic stem (ES) cells, an ES cell line may be employed, or
embryonic cells may be obtained freshly from a host, e.g. mouse, rat,
guinea pig, etc. Such cells are grown on an appropriate fibroblast-feeder
layer or grown in the presence of leukemia inhibiting factor (LIF). When
ES or embryonic cells have been transformed, they may be used to produce
transgenic animals. After transformation, the cells are plated onto a
feeder layer in an appropriate medium. Cells containing the construct may
be detected by employing a selective medium. After sufficient time for
colonies to grow, they are picked and analyzed for the occurrence of
homologous recombination or integration of the construct. Those colonies
that are positive may then be used for embryo manipulation and blastocyst
injection. Blastocysts are obtained from 4 to 6 week old superovulated
females. The ES cells are trypsinized, and the modified cells are
injected into the blastocoel of the blastocyst. After injection, the
blastocysts are returned to each uterine horn of pseudopregnant females.
Females are then allowed to go to term and the resulting offspring
screened for the construct. By providing for a different phenotype of the
blastocyst and the genetically modified cells, chimeric progeny can be
readily detected.

[0101]The chimeric animals are screened for the presence of the modified
gene and males and females having the modification are mated to produce
homozygous progeny. If the gene alterations cause lethality at some point
in development, tissues or organs can be maintained as allogeneic or
congenic grafts or transplants, or in in vitro culture. The transgenic
animals may be any non-human mammal, such as laboratory animals, domestic
animals, etc. The transgenic animals may be used in functional studies,
drug screening, etc., e.g. to determine the effect of a candidate drug on
fusion protein activity.

[0102]Also of interest are assays involving xenotransplants of
hyperproliferative cells and cell lines into immunocompromised host
animals, e.g., mice, where cellular, e.g., tumor, growth is identified as
a readout of the inhibition of the fusion protein by a candidate agent
being screened.

[0103]Typical in vivo cancer models for solid tumors involve the grafting
of a piece of primary human tumor or, more frequently, of cells from a
human tumor cell line onto immunocompromised mice like nude, SCID or
NOD/SCID mice. The human tumor cells engraft, either subcutaneously or in
a target organ. If the engraftment takes place subcutaneously, tumor
growth can be followed and recorded based on size. In order to
demonstrate activity of an anti-cancer compound, the engrafted mice are
treated with the compound and the reduction in size of the tumor is
recorded and/or the prolongation of the survival of the mice is followed.

[0104]Assays of interest for fluid or liquid tumors, e.g., leukemias,
include the following:

[0105]1) Subcutaneous injection of a liquid tumor cell line into an
immunocompromised host animal, e.g., NOD/SCID mice. Although being a
liquid tumor, the tumor cells can form a solid tumor subcutaneously in an
immunocompromised host. Once the solid tumor has reached a certain size,
administration of the candidate agent starts and activity of the
candidate agent is measured in reduction of tumor size and survival.

[0106]2) Injection of a cell line (e.g., BaF3 based) expressing the fusion
kinase into a host animal, e.g., into the tail vein of a suitable
syngeneic mouse (Balb c). The injected cells proliferate in the blood of
the animal, accumulate in the spleen and eventually kill the host animal
due to organ damage. The weight of the spleen and the time of survival
following administration of a given candidate agent serves as an
indicator for the success of the therapeutic to affect the cancer.

[0107]3) Transduction of bone marrow stem cells with a retrovirus that
leads to the expression of mutant kinase. As a result of the expression
of the mutant kinase in hematopoietic stem cells, the cells proliferate
in an unregulated way which eventually kills the host animal, e.g.,
mouse, in which they are present. A candidate agent that inhibits the
transduced activated kinase and prolongs the survival of the host animal
is one that exhibits activity against the target fusion oncokinase
polypeptide.

[0108]Whether the format is in vivo or in vitro, the model being employed
is combined with the candidate agent and the effect of the candidate
agent on the model is observed and related to the modulatory activity of
the agent being tested. For example, for screening inhibitory agents, the
model is combined with the candidate agent in an environment in which, in
the absence of the candidate agent, kinase activity is observed. The
conditions may be set up in vitro by combining the various required
components in an aqueous medium, or the assay may be carried out in vivo,
etc.

[0109]A variety of different candidate agents may be screened by the above
methods. Candidate agents encompass numerous chemical classes, though
typically they are organic molecules, preferably small organic compounds
having a molecular weight of more than 50 and less than about 2,500
daltons. Candidate agents comprise functional groups necessary for
structural interaction with proteins, particularly hydrogen bonding, and
typically include at least an amine, carbonyl, hydroxyl or carboxyl
group, preferably at least two of the functional chemical groups. The
candidate agents often comprise cyclical carbon or heterocyclic
structures and/or aromatic or polyaromatic structures substituted with
one or more of the above functional groups. A preferred class of
candidate agents includes those that mimic ATP, the co-substrate for the
phosphoryl transfer reaction catalyzed by the fusion proteins. Candidate
agents are also found among biomolecules including peptides, saccharides,
fatty acids, steroids, purines, pyrimidines, derivatives, structural
analogs or combinations thereof.

[0110]Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a wide
variety of organic compounds and biomolecules, including expression of
randomized oligonucleotides and oligopeptides. Alternatively, libraries
of natural compounds in the form of bacterial, fungal, plant and animal
extracts are available or readily produced. Additionally, natural
synthetically produced libraries and compounds are readily modified
through conventional chemical, physical and biochemical means, and may be
used to produce combinatorial libraries. Known pharmacological agents may
be subjected to directed or random chemical modifications, such as
acylation, alkylation, esterification, amidification, etc. to produce
structural analogs.

[0111]Agents identified in the above screening assays that inhibit
activity of the subject fusion proteins find use in various methods,
where representative methods are described below.

Methods of Modulating Fusion Protein Activity

[0112]Also provided by the subject invention are methods of modulating,
including enhancing and repressing, the activity of the subject fusion
proteins. As such, methods of both increasing and decreasing fusion
protein kinase activity are provided. In many embodiments, such methods
are methods of inhibiting fusion protein kinase activity.

[0113]One representative method of inhibiting fusion protein activity is
to employ small molecules that inhibit the fusion protein activity.
Naturally occurring or synthetic small molecule compounds of interest
include numerous chemical classes, though typically they are organic
molecules, preferably small organic compounds having a molecular weight
of more than 50 and less than about 2,500 daltons. Candidate agents
comprise functional groups necessary for structural interaction with
proteins, particularly hydrogen bonding, and typically include at least
an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two
of the functional chemical groups. The candidate agents often comprise
cyclical carbon or heterocyclic structures and/or aromatic or
polyaromatic structures substituted with one or more of the above
functional groups. Candidate agents are also found among biomolecules
including peptides, saccharides, fatty acids, steroids, purines,
pyrimidines, derivatives, structural analogs (especially of ATP) or
combinations thereof. Such molecules may be identified, among other ways,
by employing the screening protocols described above.

[0114]In yet other embodiments, expression of the target fusion protein is
inhibited. Inhibition of target fusion protein expression may be
accomplished using any convenient means, including administration of an
agent that inhibits target fusion protein expression (e.g., antisense
agents), inactivation of the encoding gene, e.g., through recombinant
techniques, etc.

[0115]Antisense molecules can be used to down-regulate expression of the
target protein in cells. The anti-sense reagent may be antisense
oligonucleotides (ODN), particularly synthetic ODN having chemical
modifications from native nucleic acids, or nucleic acid constructs that
express such anti-sense molecules as RNA. The antisense sequence is
complementary to the mRNA of the targeted gene, and inhibits expression
of the targeted gene products. Antisense molecules inhibit gene
expression through various mechanisms, e.g., by reducing the amount of
mRNA available for translation, through activation of RNAse H, or steric
hindrance. One or a combination of antisense molecules may be
administered, where a combination may comprise multiple different
sequences.

[0116]Antisense molecules may be produced by expression of all or a part
of the target gene sequence in an appropriate vector, where the
transcriptional initiation is oriented such that an antisense strand is
produced as an RNA molecule. Alternatively, the antisense molecule is a
synthetic oligonucleotide. Antisense oligonucleotides will generally be
at least about 7, usually at least about 12, more usually at least about
20 nucleotides in length, and not more than about 500, usually not more
than about 50, more usually not more than about 35 nucleotides in length,
where the length is governed by efficiency of inhibition, specificity,
including absence of cross-reactivity, and the like. It has been found
that short oligonucleotides, of from 7 to 8 bases in length, can be
strong and selective inhibitors of gene expression (see Wagner et al.
(1996), Nature Biotechnol. 14:840-844).

[0117]A specific region or regions of the endogenous sense strand mRNA
sequence is chosen to be complemented by the antisense sequence.
Selection of a specific sequence for the oligonucleotide may use an
empirical method, where several candidate sequences are assayed for
inhibition of expression of the target gene in an in vitro or animal
model. A combination of sequences may also be used, where several regions
of the mRNA sequence are selected for antisense complementation.

[0118]Antisense oligonucleotides may be chemically synthesized by methods
known in the art (see Wagner et al. (1993), supra, and Milligan et al.,
supra.) Preferred oligonucleotides are chemically modified from the
native phosphodiester structure, in order to increase their intracellular
stability and binding affinity. A number of such modifications have been
described in the literature, which alter the chemistry of the backbone,
sugars or heterocyclic bases.

[0119]Among useful changes in the backbone chemistry are
phosphorothioates; phosphorodithioates, where both of the non-bridging
oxygens are substituted with sulfur; phosphoroamidites; alkyl
phosphotriesters and boranophosphates. Achiral phosphate derivatives
include 3'-O'-5'-S-phosphorothioate, 3'-S-5'-O-phosphorothioate,
3'-CH2-5'-O-phosphonate and 3'-NH-5'-O-phosphoroamidate. Peptide
nucleic acids replace the entire ribose phosphodiester backbone with a
peptide linkage. Sugar modifications are also used to enhance stability
and affinity. The α-anomer of deoxyribose may be used, where the
base is inverted with respect to the natural β-anomer. The 2'-OH of
the ribose sugar may be altered to form 2'-O-methyl or 2'-O-allyl sugars,
which provides resistance to degradation without comprising affinity.
Modification of the heterocyclic bases must maintain proper base pairing.
Some useful substitutions include deoxyuridine for deoxythymidine;
5-methyl-2'-deoxycytidine and 5-bromo-2'-deoxycytidine for deoxycytidine.
5-propynyl-2'-deoxyuridine and 5-propynyl-2'-deoxycytidine have been
shown to increase affinity and biological activity when substituted for
deoxythymidine and deoxycytidine, respectively.

[0120]As an alternative to anti-sense inhibitors, catalytic nucleic acid
compounds, e.g. ribozymes, anti-sense conjugates, etc. may be used to
inhibit gene expression. Ribozymes may be synthesized in vitro and
administered to the patient, or may be encoded on an expression vector,
from which the ribozyme is synthesized in the targeted cell (for example,
see International Patent Application WO 95/23225, and Beigelman et al.
(1995), Nucl. Acids Res. 23:4434-42). Examples of oligonucleotides with
catalytic activity are described in WO 95/06764. Conjugates of anti-sense
ODN with a metal complex, e.g. terpyridylCu(II), capable of mediating
mRNA hydrolysis are described in Bashkin et al. (1995), Appl. Biochem.
Biotechnol. 54:43-56.

[0121]In another embodiment, the target protein gene is inactivated so
that it no longer expresses the target fusion protein. By inactivated is
meant that the gene, e.g., coding sequence and/or regulatory elements
thereof, is genetically modified so that it no longer expresses a
protein, or at least a functional protein. The alteration or mutation may
take a number of different forms, e.g., through deletion of one or more
nucleotide residues in the fusion protein region, through exchange of one
or more nucleotide residues in the fusion protein region, and the like.
One means of making such alterations in the coding sequence is by
homologus recombination. Methods for generating targeted gene
modifications through homologous recombination are known in the art,
including those described in: U.S. Pat. Nos. 6,074,853; 5,998,209;
5,998,144; 5,948,653; 5,925,544; 5,830,698; 5,780,296; 5,776,744;
5,721,367; 5,614,396; 5,612,205; the disclosures of which are herein
incorporated by reference.

[0122]The above-described methods of inhibiting fusion protein activity
find use in a number of different applications. In many applications, the
subject methods and compositions are employed to inhibit fusion protein
activity in a cell that endogenously comprises a coding sequence for the
target fusion protein. Expression of the target gene is considered to be
inhibited if, consistent with the above description, expression is
decreased by at least about 2 fold, usually at least about 5 fold and
often by at least about 25, about 50, about 100 fold or more, as compared
to a control, e.g., an otherwise identical cell not subjected to the
subject methods.

[0123]A more specific application in which the subject methods find use is
to decrease the proliferative capacity of a cell. The term "proliferative
capacity" as used herein refers to the number of divisions that a cell
can undergo, and preferably to the ability of the target cell to continue
to divide. The subject methods typically result in a decrease in
proliferative capacity of at least about 1.2-2 fold, usually at least
about 5 fold and often at least about 10, 20, 50 fold or even higher,
compared to a control.

[0124]Another specific application in which the subject methods find use
is to induce apoptosis, or programmed cell death, in a cell. The subject
methods typically result in a decrease in the viable cell counts of at
least 20%, usually at least 50%, and often of at least 90% or even
higher.

Therapeutic Applications of Fusion Protein Activity Modulation

[0125]The methods also find use in a variety of therapeutic applications
in which it is desired to modulate, e.g., increase or decrease, and
typically decrease, fusion protein kinase activity in a target cell or
collection of cells, where the collection of cells may be a whole animal
or portion thereof, e.g., tissue, organ, etc. As such, the target cell(s)
may be a host animal or portion thereof. In such methods, an effective
amount of an active agent that modulates fusion protein activity, e.g.,
enhances or decreases oncokinase activity as desired, is administered to
the target cell or cells, e.g., by contacting the cells with the agent,
by administering the agent to the animal, etc. By effective amount is
meant a dosage sufficient to modulate fusion protein activity in the
target cell(s), as desired.

[0126]A variety of different types of agents may be employed, including
the representative types of candidate agents described above, e.g., small
molecule agents, nucleic acid agents, polypeptide agents, etc.

[0127]In certain embodiments, the agents are pyrimidine derivatives as
described in U.S. Pat. No. 5,521,184, the disclosure of which is herein
incorporated by reference. In these embodiments, of interest are
N-phenyl-2-pyrimidine-amine derivatives of formula (I):

##STR00001##

[0128]wherein

[0129]R1 is 4-pyrazinyl, 1-methyl-1H-pyrrolyl, amino- or amino-lower
alkyl-substituted phenyl wherein the amino group in each case is free,
alkylated or acylated, 1H-indolyl or 1H-imidazolyl bonded at a
five-membered ring carbon atom, or unsubstituted or lower
alkyl-substituted pyridyl bonded at a ring carbon atom and unsubstituted
or substituted at the nitrogen atom by oxygen,

[0130]R2 and R3 are each independently of the other hydrogen or
lower alkyl,

[0131]one or two of the radicals R4, R5, R6, R7 and
R8 are each nitro, fluoro-substituted lower alkoxy or a radical of
formula (II):

[0136]R10 is an aliphatic radical having at least 5 carbon atoms, or
an aromatic, aromatic-aliphatic, cycloaliphatic,
cycloaliphatic-aliphatic, heterocyclic or heterocyclic-aliphatic radical,

[0137]and the remaining radicals R4, R5, R6, R7 and
R8; are each independently of the others hydrogen, lower alkyl that
is unsubstituted or substituted by free or alkylated amino, piperazinyl,
piperidinyl, pyrrolidinyl or by morpholinyl, or lower alkanoyl,
trifluoromethyl, free, etherified or esterifed hydroxy, free, alkylated
or acylated amino or free or esterified carboxy,

[0138]and salts of such compounds having at least one salt-forming group.

[0139]In these embodiments:

[0140]1-Methyl-1H-pyrrolyl is preferably 1-methyl-1H-pyrrol-2-yl or
1-methyl-1H-pyrrol-3-yl.

[0141]Amino- or amino-lower alkyl-substituted phenyl R1 wherein the
amino group in each case is free, alkylated or acylated, is phenyl
substituted in any desired position (ortho, meta or para) wherein an
alkylated amino group is preferably mono- or di-lower alkylamino, for
example dimethylamino, and the lower alkyl moiety of amino-lower alkyl is
preferably linear C1-C3 alkyl, such as especially methyl or
ethyl.

[0142]1H-Indolyl bonded at a carbon atom of the five-membered ring is
1H-indol-2-yl or 1H-indol-3-yl.

[0143]Unsubstituted or lower alkyl-substituted pyridyl bonded at a ring
carbon atom is lower alkyl-substituted or preferably unsubstituted 2-, or
preferably 3- or 4-pyridyl, for example 3-pyridyl, 2-methyl-3-pyridyl,
4-methyl-3-pyridyl or 4-pyridyl. Pyridyl substituted at the nitrogen atom
by oxygen is a radical derived from pyridine N-oxide, i.e.
N-oxido-pyridyl, e.g. N-oxido-4-pyridyl.

[0148]The term "lower" within the scope of this text denotes radicals
having up to and including 7, preferably up to and including 4 carbon
atoms.

[0149]Lower alkyl R1, R2, R3 and R9 is preferably
methyl or ethyl.

[0150]An aliphatic radical R10 having at least 5 carbon atoms
preferably has not more than 22 carbon atoms, generally not more than 10
carbon atoms, and is such a substituted or preferably unsubstituted
aliphatic hydrocarbon radical, that is to say such a substituted or
preferably unsubstituted alkynyl, alkenyl or preferably alkyl radical,
such as C5-C7 alkyl, for example n-pentyl. An aromatic radical
R10 has up to 20 carbon atoms and is unsubstituted or substituted,
for example in each case unsubstituted or substituted naphthyl, such as
especially 2-naphthyl, or preferably phenyl, the substituents preferably
being selected from cyano, unsubstituted or hydroxy-, amino- or
4-methyl-piperazinyl-substituted lower alkyl, such as especially methyl,
trifluoromethyl, free, etherified or esterified hydroxy, free, alkylated
or acylated amino and free or esterified carboxy. In an
aromatic-aliphatic radical R10 the aromatic moiety is as defined
above and the aliphatic moiety is preferably lower alkyl, such as
especially C1-C2 alkyl, which is substituted or preferably
unsubstituted, for example benzyl. A cycloaliphatic radical R10 has
especially up to 30, more especially up to 20, and most especially up to
10 carbon atoms, is mono- or poly-cyclic and is substituted or preferably
unsubstituted, for example such a cycloalkyl radical, especially such a
5- or 6-membered cycloalkyl radical, such as preferably cyclohexyl. In a
cycloaliphatic-aliphatic radical R10 the cycloaliphatic moiety is as
defined above and the aliphatic moiety is preferably lower alkyl, such as
especially C1-C2 alkyl, which is substituted or preferably
unsubstituted. A heterocyclic radical R10 contains especially up to
20 carbon atoms and is preferably a saturated or unsaturated monocyclic
radical having 5 or 6 ring members and 1-3 hetero atoms which are
preferably selected from nitrogen, oxygen and sulfur, especially, for
example, thienyl or 2-, 3- or 4-pyridyl, or a bi- or tri-cyclic radical
wherein, for example, one or two benzene radicals are annellated (fused)
to the mentioned monocyclic radical. In a heterocyclic-aliphatic radical
R10 the heterocyclic moiety is as defined above and the aliphatic
moiety is preferably lower alkyl, such as especially C1-C2
alkyl, which is substituted or preferably unsubstituted.

[0151]Etherified hydroxy is preferably lower alkoxy. Esterified hydroxy is
preferably hydroxy esterified by an organic carboxylic acid, such as a
lower alkanoic acid, or a mineral acid, such as a hydrohalic acid, for
example lower alkanoyloxy or especially halogen, such as iodine, bromine
or especially fluorine or chlorine.

[0152]Alkylated amino is, for example, lower alkylamino, such as
methylamino, or di-lower alkylamino, such as dimethylamino. Acylated
amino is, for example, lower alkanoylamino or benzoylamino.

[0153]Esterified carboxy is, for example, lower alkoxycarbonyl, such as
methoxycarbonyl.

[0154]A substituted phenyl radical may carry up to 5 substituents, such as
fluorine, but especially in the case of relatively large substituents is
generally substituted by only from 1 to 3 substituents. Examples of
substituted phenyl that may be given special mention are 4-chloro-phenyl,
pentafluoro-phenyl, 2-carboxy-phenyl, 2-methoxy-phenyl, 4-fluorophenyl,
4-cyano-phenyl and 4-methyl-phenyl.

[0155]Salt-forming groups in a compound of formula (I) are groups or
radicals having basic or acidic properties. Compounds having at least one
basic group or at least one basic radical, for example a free amino
group, a pyrazinyl radical or a pyridyl radical, may form acid addition
salts, for example with inorganic acids, such as hydrochloric acid,
sulfuric acid or a phosphoric acid, or with suitable organic carboxylic
or sulfonic acids, for example aliphatic mono- or di-carboxylic acids,
such as trifluoroacetic acid, acetic acid, propionic acid, glycolic acid,
succinic acid, maleic acid, fumaric acid, hydroxymaleic acid, malic acid,
tartaric acid, citric acid or oxalic acid, or amino acids such as
arginine or lysine, aromatic carboxylic acids, such as benzoic acid,
2-phenoxy-benzoic acid, 2-acetoxybenzoic acid, salicylic acid,
4-aminosalicylic acid, aromatic-aliphatic carboxylic acids, such as
mandelic acid or cinnamic acid, heteroaromatic carboxylic acids, such as
nicotinic acid or isonicotinic acid, aliphatic sulfonic acids, such as
methane-, ethane- or 2-hydroxyethane-sulfonic acid, or aromatic sulfonic
acids, for example benzene-, p-toluene- or naphthalene-2-sulfonic acid.
When several basic groups are present mono- or poly-acid addition salts
may be formed.

[0156]Compounds of formula (I) having acidic groups, for example a free
carboxy group in the radical R10, may form metal or ammonium salts,
such as alkali metal or alkaline earth metal salts, for example sodium,
potassium, magnesium or calcium salts, or ammonium salts with ammonia or
suitable organic amines, such as tertiary monoamines, for example
triethylamine or tri-(2-hydroxyethyl)-amine, or heterocyclic bases, for
example N-ethylpiperidine or N,N'-dimethyl-piperazine. Compounds of
formula (I) having both acidic and basic groups can form internal
salts--

[0157]Of particular interest in these embodiments is a pyrimidine
derivative described in this patent in which R1 is 3-pyridyl,
R2, R3, R5, R6, and R8 are each hydrogen,
R4 is methyl, and R7 is a group of formula (II) in which
R9 is hydrogen, X is oxo, k is 0, and R10 is
4-[(4-methyl-1-piperazinyl)methyl]phenyl. The mesylate salt of this
compound having the chemical name
4-[(4-methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyri-
midinyl]amino-phenyl]benzamide methanesulfonate is now commonly known as
imatinib mesylate and sold under the trademark Gleevec®

[0158]In yet other embodiments of interest, the agent is not imatinib
mesylate.

[0159]Also of interest are phthalazine compounds of formula (III):

##STR00002##

wherein r is 0 to 2, n is 0 to 2; m is 0 to 4; R11 and R12 (i)
are in each case a lower alkyl, or (ii) together form a bridge in
subformula (III*)

##STR00003##

or (iii) together form a bridge in subformula (III**):

##STR00004##

wherein one or two of the ring members Ti, T2, T3, and
T4 are nitrogen, and the remainder are in each case CH; A, B, D, and
E are N or CH, wherein not more than 2 of these radicals are N; G is
lower alkylene, acyloxy- or hydroxy-lower alkylene, --CH2--O--,
--CH2--S--, --CH2--NH--, oxa, thia, or imino; Q is lower alkyl,
especially methyl; R is H or lower alkyl; X is imino, oxa, or thia; Y is
aryl, pyridyl, or (un)substituted cycloalkyl; and Z is independently
mono- or disubstituted amino, halogen, alkyl, substituted alkyl, hydroxy,
etherified or esterified hydroxy, nitro, cyano, carboxy, esterified
carboxy, alkanoyl, carbamoyl, N-mono- or N,N-disubstituted carbamoyl,
amidino, guanidino, mercapto, sulfo, phenylthio, phenyl-lower alkylthio,
alkylphenylthio, phenylsulfinyl, phenyl-lower alkylsulfinyl,
alkylphenylsulfinyl, phenylsulfonyl, phenyl-lower alkylsulfonyl, or
alkylphenylsulfonyl; and wherein the dashed lines independently represent
optional double bonds; or an N-oxide of said compound with the
stipulation that, if Y is pyridyl or unsubstituted cycloalkyl, X is
imino, and the remaining radicals are as defined, then G is selected from
the group comprising lower alkylene, --CH2--O--, --CH2--S--,
oxa and thia; or a salt thereof. Such compounds, e.g., PTK787 (also known
as Vatalanib), are further described in WO 98/35958, U.S. patent
application Ser. No. 09/859,858, and U.S. Pat. No. 6,258,812 B1; the
disclosure of the latter of which is herein incorporated by reference.

[0160]Also of interest in certain embodiments are the protein tyrosine
kinase inhibitors of formula (IV):

##STR00005##

in which:

[0161](i) R13 represents a hydrogen atom or a C1-4alkyl group;
and R14 represents a group of formula -A1-NR17R19 in
which each of R17 and R18 independently represents a hydrogen
atom or a C1-4alkyl group and A1 represents (CH2)m',
(CH2)n'-A2-(CH2)p', or
(CH2CH2O)q'CH2CH2 in which m' is an integer of
from 2 to 10, each of n' and p' is an integer of from 1 to 6, A2 is
CH═CH, phenylene, biphenylene, cyclohexylene or piperazinylene and q'
is 1, 2 or 3;

[0162](ii) R13 and R14 together represent
-A3-NR19-A4- in which each of A3 and A4
independently represents (CH2)r' or
(CH2CH2O)s'CH2CH2 in which r' is an integer of
from 2 to 6, s' is 1, 2 or 3, and R19 represents a hydrogen atom or
a C1-4alkyl group;

[0163](iii) R13 and R14 together with the nitrogen atom to which
they are attached represent a piperidinyl group, which piperidinyl group
bears a substituent of formula -A5-R20 at the 4 position, in
which A5 represents C1-4alkylene and R20 represents
piperidin-4-yl; or

[0164](iv) R13 and R14 together with the nitrogen atom to which
they are attached represent a pyrrolidinyl, piperidinyl or morpholino
group; and

[0165]R15 and R16 each independently represents a hydrogen atom,
a halogen atom, a C1-4alkyl group, a C1-4alkoxy group, a phenyl
group which is unsubstituted or substituted by one or two substituents
selected independently from a halogen atom, a C1-4alkyl group and a
C1-4alkoxy group, a group of formula R21S(O)2NR22--,
a group of formula R23N(R24)S(O)2--, a group of formula
R25C(O)N(R26)-- or a group of formula R27N(R28)C(O)--
in which each of R21, R23, R25 and R27 independently
represents a C1-4alkyl group or a phenyl group which is
unsubstituted or substituted by one or two substituents selected
independently from a halogen atom, a C1-4alkyl group and a
C1-4alkoxy group, and each of R22, R24, R26 and
R28 independently represents a hydrogen atom or a C1-4alkyl
group;

[0166]or a pharmaceutically-acceptable salt thereof.

[0167]An inhibitor of formula (IV) of particular interest, identified as
THRX-165724, is one in which R13 and R14 and the nitrogen to
which they are attached form a piperazinyl ring and R15 and R16
are both hydrogen. Compounds of formula (IV) are described in U.S. Patent
Application Ser. Nos. 60/343,746, 60/343,813, and 10/327,385, the
disclosures of which are herein incorporated by reference.

[0168]Another group of compounds of interest in certain embodiments are
compounds of formula (V):

##STR00006##

wherein:

[0169]R29 is selected from the group consisting of --CN, --X,
--CX3, --R33, --CO2R33, --SO2R33,
--O--C1-8alkyl that is straight or branched chained, --O-phenyl,
--O-napthyl, --O-indolyl, and --O-isoquinolinyl, in which X is a halogen,
and R33 is hydrogen or a C1-8alkyl that is straight or branched
chained,

[0170]R30 and R32 are each independently selected from the group
consisting of --O--CH3, --O--CH2--CH3,
--O--CH2--CH═CH2, --O--CH2--C≡CH,
--O(CH2)--SO2--R33,
--O--CH2--CH(R34)CH2--R31 and
--O(--CH2)n'--R31, in which R34 is --OH, --X, or a
C1-8alkyl that is straight or branched chained, n'' is 2 or 3, and

[0172]Among compounds of formula (V), of particular interest is the
compound, identified as MLN518 in which R29 is --O-isopropyl,
R30 is --O--(CH2)3-piperidin-1-yl, and R32 is
--OCH3. Compounds of formula (V) are further described in WO
02/16351, which is incorporated herein by reference.

[0173]Yet another group of compounds of interest in certain embodiments
are compounds of formula (VI):

[0177]R38 is selected from the group consisting of hydrogen, halo,
alkyl, hydroxy, alkoxy, and --NR46R47;

[0178]R39 is selected from the group consisting of hydrogen, alkyl
and --C(O)R40;

[0179]R41 is selected from the group consisting of hydrogen, alkyl,
aryl, heteroaryl, --C(O)R50 and --C(O)R40;

[0180]R42 and R43 are independently selected from the group
consisting of hydrogen, alkyl and aryl;

[0181]R40 is selected from the group consisting of hydroxy, alkoxy,
aryloxy, --N(R44)(CH2)n*--R45, and
--NR46R47;

[0182]R44 is selected from the group consisting of hydrogen and
alkyl;

[0183]R45 is selected from the group consisting of
--NR46R47, hydroxy, --C(O)R48, aryl, heteroaryl,
--N.sup.+(O.sup.-)R46R47, --N(OH)R46, and --NHC(O)Ra,
wherein Ra is unsubstituted alkyl, haloalkyl, or aralkyl;

[0184]R46 and R47 are independently selected from the group
consisting of hydrogen, alkyl, lower alkyl substituted with
hydroxyalkylamino, cyanoalkyl, cycloalkyl, aryl, and heteroaryl; or

[0185]R46 and R47 may combine to form a heterocyclo group;

[0186]R48 is selected from the group consisting of hydrogen, hydroxy,
alkoxy, and aryloxy;

[0187]R49 is selected from the group consisting of hydroxy,
--C(O)R48, --NR46R47 and --C(O)NR46R47;

[0188]R50 is selected from the group consisting of alkyl, cycloalkyl,
aryl and heteroaryl; and

[0189]n* and r* are independently 1, 2, 3, or 4;

or a pharmaceutically-acceptable salt thereof.

[0190]A compound of formula (VI) of particular interest, identified as
SU11248, is the compound in which R36 is fluoro, R35, R37,
and R38 are each hydrogen, R39 and R41 are each methyl,
and R40 is --N(H)(CH2)2N(C2H5)2. Compounds
of formula (VI) are described in WO 01/60814, the disclosure of which is
incorporated herein by reference.

[0191]In one embodiment, the agent employed in the methods of this
invention is selected from the group consisting of:

##STR00008##

[0192]or a pharmaceutically-acceptable salt thereof.

[0193]Also of interest are other protein tyrosine kinase inhibitors. Such
inhibitors include, but are not limited to, the tyrosine kinase
inhibitors appearing in Appendix A of the priority provisional
applications having Ser. Nos. 60/402,330 filed on Aug. 9, 2002 and
60/440,491 filed on Jan. 16, 2003; the disclosures of which are herein
incorporated by reference.

[0194]In the subject methods, the active agent(s) may be administered to
the targeted cells using any convenient means capable of resulting in the
desired modulation of fusion protein activity. Thus, the agent can be
incorporated into a variety of formulations for therapeutic
administration. More particularly, the agents of the present invention
can be formulated into pharmaceutical compositions by combination with
appropriate, pharmaceutically acceptable carriers or diluents, and may be
formulated into preparations in solid, semi-solid, liquid or gaseous
forms, such as tablets, capsules, powders, granules, ointments,
solutions, suppositories, injections, inhalants and aerosols. As such,
administration of the agents can be achieved in various ways, including
oral, buccal, rectal, parenteral, intraperitoneal, intradermal,
transdermal, intratracheal, etc., administration.

[0195]In pharmaceutical dosage forms, the agents may be administered in
the form of their pharmaceutically acceptable salts, or they may also be
used alone or in appropriate association, as well as in combination, with
other pharmaceutically active compounds. The following methods and
excipients are merely exemplary and are in no way limiting.

[0196]For oral preparations, the agents can be used alone or in
combination with appropriate additives to make tablets, powders, granules
or capsules, for example, with conventional additives, such as lactose,
mannitol, corn starch or potato starch; with binders, such as crystalline
cellulose, cellulose derivatives, acacia, corn starch or gelatins; with
disintegrators, such as corn starch, potato starch or sodium
carboxymethylcellulose; with lubricants, such as talc or magnesium
stearate; and if desired, with diluents, buffering agents, moistening
agents, preservatives and flavoring agents.

[0197]The agents can be formulated into preparations for injection by
dissolving, suspending or emulsifying them in an aqueous or nonaqueous
solvent, such as vegetable or other similar oils, synthetic aliphatic
acid glycerides, esters of higher aliphatic acids or propylene glycol;
and if desired, with conventional additives such as solubilizers,
isotonic agents, suspending agents, emulsifying agents, stabilizers and
preservatives.

[0198]The agents can be utilized in aerosol formulation to be administered
via inhalation. The compounds of the present invention can be formulated
into pressurized acceptable propellants such as dichlorodifluoromethane,
propane, nitrogen and the like.

[0199]Furthermore, the agents can be made into suppositories by mixing
with a variety of bases such as emulsifying bases or water-soluble bases.
The compounds of the present invention can be administered rectally via a
suppository. The suppository can include vehicles such as cocoa butter,
carbowaxes and polyethylene glycols, which melt at body temperature, yet
are solidified at room temperature.

[0200]Unit dosage forms for oral or rectal administration such as syrups,
elixirs, and suspensions may be provided wherein each dosage unit, for
example, teaspoonful, tablespoonful, tablet or suppository, contains a
predetermined amount of the composition containing one or more
inhibitors. Similarly, unit dosage forms for injection or intravenous
administration may comprise the inhibitor(s) in a composition as a
solution in sterile water, normal saline or another pharmaceutically
acceptable carrier.

[0201]The term "unit dosage form," as used herein, refers to physically
discrete units suitable as unitary dosages for human and animal subjects,
each unit containing a predetermined quantity of compounds of the present
invention calculated in an amount sufficient to produce the desired
effect in association with a pharmaceutically acceptable diluent, carrier
or vehicle. The specifications for the novel unit dosage forms of the
present invention depend on the particular compound employed and the
effect to be achieved, and the pharmacodynamics associated with each
compound in the host.

[0202]Where the agent of interest is imatinib mesylate, the dosage
employed in certain embodiments is substantially less than that which is
employed for the use of imatinib mesylate in the treatment of chronic
myeloid leukemia (CML). By substantially less is meant at least about
2-fold, usually at least about 3-fold and more usually at least about
4-fold less than the dosages employed in the treatment of CML, where
typical dosages of imatinib mesylate employed in the subject methods may
range from about 30 mg/day to about 300 mg/day, usually from about 50
mg/day to about 200 mg/day. In yet other embodiments, the dosage employed
is the same as or greater than that employed in the treatment of CML. The
pharmaceutically acceptable excipients, such as vehicles, adjuvants,
carriers or diluents, are readily available to the public. Moreover,
pharmaceutically acceptable auxiliary substances, such as pH adjusting
and buffering agents, tonicity adjusting agents, stabilizers, wetting
agents and the like, are readily available to the public.

[0203]The invention further provdes a process for producing a
pharmaceutical composition comprising a pharmaceutically acceptable
carrier and an agent that inhibits the activity of an oncokinase fusion
polypeptide of the invention. The process comprises admixing a
pharmaceutically acceptable carrier with an agent wherein the agent is
identified by a screening method comprising (1) contacting an oncokinase
fusion polypeptide of the invention with test agent, and determining the
effect, if any, of the test agent on the activity of the fusion
polypeptide; or (2) contacting a coding sequence for a fusion polypeptide
of the invention with a test agent, and determining the effect, if any,
of the test agent on the expression of the fusion polypeptide from the
coding sequence.

[0204]Where the agent is a polypeptide, polynucleotide, analog or mimetic
thereof, e.g. an antisense molecule, it may be introduced into tissues or
host cells by any number of routes, including viral infection,
microinjection, or fusion of vesicles. Jet injection may also be used for
intramuscular administration, as described by Furth et al. (1992), Anal
Biochem 205:365-368. The DNA may be coated onto gold microparticles, and
delivered intradermally by a particle bombardment device, or "gene gun"
as described in the literature (see, for example, Tang et al. (1992),
Nature 356:152-154), where gold microprojectiles are coated with the DNA,
then bombarded into skin cells. For nucleic acid therapeutic agents, a
number of different delivery vehicles find use, including viral and
non-viral vector systems, as are known in the art.

[0205]Those of skill in the art will readily appreciate that dose levels
can vary as a function of the specific compound, the nature of the
delivery vehicle, and the like. Preferred dosages for a given compound
are readily determinable by those of skill in the art by a variety of
means.

[0206]The subject methods find use in the treatment of a variety of
different conditions in which the modulation of the subject oncokinase
fusion protein activity in the host is desired. By treatment is meant
that at least an amelioration of the symptoms associated with the
condition afflicting the host is achieved, where amelioration is used in
a broad sense to refer to at least a reduction in the magnitude of a
parameter, e.g. symptom, associated with the condition being treated. As
such, treatment also includes situations where the pathological
condition, or at least symptoms associated therewith, are completely
inhibited, e.g., prevented from happening, or stopped, e.g. terminated,
such that the host no longer suffers from the condition, or at least the
symptoms that characterize the condition.

[0207]A variety of hosts are treatable according to the subject methods.
Generally such hosts are "mammals" or "mammalian," where these terms are
used broadly to describe organisms which are within the class mammalia,
including the orders carnivore (e.g., dogs and cats), rodentia (e.g.,
mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees,
and monkeys). In many embodiments, the hosts will be humans.

[0208]Methods for inhibiting fusion protein activity according to the
subject invention find use in, among other applications, the treatment of
cellular proliferative disease conditions, including neoplastic disease
conditions, i.e., cancers. In such applications, an effective amount of
an active agent, e.g., an agent that inhibits fusion protein activity, is
administered to the subject in need thereof. Treatment is used broadly as
defined above, e.g., to include at least an amelioration in one or more
of the symptoms of the disease, as well as a complete cessation thereof,
as well as a reversal and/or complete removal of the disease condition,
e.g., cure.

[0209]There are many disorders associated with a dysregulation of cellular
proliferation, i.e., cellular hyperproliferative disorders. The
conditions of interest include, but are not limited to, the following
conditions.

[0210]The subject methods may be employed in the treatment of a variety of
conditions where there is proliferation and/or migration of smooth muscle
cells, and/or inflammatory cells into the intimal layer of a vessel,
resulting in restricted blood flow through that vessel, i.e. neointimal
occlusive lesions. Occlusive vascular conditions of interest include
atherosclerosis, graft coronary vascular disease after transplantation,
vein graft stenosis, peri-anastomatic prosthetic graft stenosis,
restenosis after angioplasty or stent placement, and the like.

[0211]Diseases where there is hyperproliferation and tissue remodelling or
repair of reproductive tissue, e.g. uterine, testicular and ovarian
carcinomas, endometriosis, squamous and glandular epithelial carcinomas
of the cervix, etc. are reduced in cell number by administration of the
subject compounds

[0213]Some cancers of particular interest include breast cancers, which
are primarily adenocarcinoma subtypes. Ductal carcinoma in situ (DCIS) is
the most common type of noninvasive breast cancer. In DCIS, the malignant
cells have not metastasized through the walls of the ducts into the fatty
tissue of the breast. Infiltrating (or invasive) ductal carcinoma (IDC)
has metastasized through the wall of the duct and invaded the fatty
tissue of the breast. Infiltrating (or invasive) lobular carcinoma (ILC)
is similar to IDC, in that it has the potential to metastasize elsewhere
in the body. About 10% to 15% of invasive breast cancers are invasive
lobular carcinomas.

[0214]Also of interest is non-small cell lung carcinoma. Non-small cell
lung cancer (NSCLC) is made up of three general subtypes of lung cancer.
Epidermoid carcinoma (also called squamous cell carcinoma) usually starts
in one of the larger bronchial tubes and grows relatively slowly. The
size of these tumors can range from very small to quite large.
Adenocarcinoma starts growing near the outside surface of the lung and
may vary in both size and growth rate. Some slowly growing
adenocarcinomas are described as alveolar cell cancer. Large cell
carcinoma starts near the surface of the lung, grows rapidly, and the
growth is usually fairly large when diagnosed. Other less common forms of
lung cancer are carcinoid, cylindroma, mucoepidermoid, and malignant
mesothelioma.

[0215]Melanoma is a malignant tumor of melanocytes. Although most
melanomas arise in the skin, they also may arise from mucosal surfaces or
at other sites to which neural crest cells migrate. Melanoma occurs
predominantly in adults, and more than half of the cases arise in
apparently normal areas of the skin. Prognosis is affected by clinical
and histological factors and by anatomic location of the lesion.
Thickness and/or level of invasion of the melanoma, mitotic index, tumor
infiltrating lymphocytes, and ulceration or bleeding at the primary site
affect the prognosis. Clinical staging is based on whether the tumor has
spread to regional lymph nodes or distant sites. For disease clinically
confined to the primary site, the greater the thickness and depth of
local invasion of the melanoma, the higher the chance of lymph node
metastases and the worse the prognosis. Melanoma can spread by local
extension (through lymphatics) and/or by hematogenous routes to distant
sites. Any organ may be involved by metastases, but lungs and liver are
common sites.

[0216]Other hyperproliferative diseases of interest relate to epidermal
hyperproliferation, tissue remodelling and repair. For example, the
chronic skin inflammation of psoriasis is associated with hyperplastic
epidermal keratinocytes as well as infiltrating mononuclear cells,
including CD4+ memory T cells, neutrophils and macrophages.

[0217]The proliferation of immune cells is associated with a number of
autoimmune and lymphoproliferative disorders. Diseases of interest
include multiple sclerosis, rheumatoid arthritis and insulin dependent
diabetes mellitus. Evidence suggests that abnormalities in apoptosis play
a part in the pathogenesis of systemic lupus erythematosus (SLE). Other
lymphoproliferative conditions include the inherited disorder of
lymphocyte apoptosis, which is an autoimmune lymphoproliferative
syndrome, as well as a number of leukemias and lymphomas. Symptoms of
allergies to environmental and food agents, as well as inflammatory bowel
disease, may also be alleviated by the compounds of the invention.

[0218]One hyperproliferative disorder of particular interest is
hypereosinophilic disorders, e.g., hypereosinophilia syndrome or HES.
Patients with hypereosinophilic syndrome (HES) present with persistent
high eosinophilic cell counts. In the course of the disease acute myeloid
eosinophilic leukemia often develops. The underlying cause for HES is not
known. The disease is highly lethal because of significant end-organ
damage. For a review of this particular disease of interest, see Bain et
al., Curr. Opin. Hematol. (2000) 7:21-25.

[0220]Also provided by the subject methods are pharmacogenomic therapeutic
methods. In these pharmacogenomic applications or methods, a
subject/host/patient is first diagnosed for the presence or absence of
the subject fusion proteins or coding sequences therefore, e.g., using a
diagnostic protocol such as those diagnostic protocols described above.
The subject is then treated using a pharmacological protocol, where the
suitability of the protocol for a particular subject/patient is
determined using the results of the diagnosis step. As such, the subject
invention provides methods of rational therapeutic protocol
determination.

[0221]For example, where the diagnosis results in the determination that
the host suffers from a disease condition characterized by the presence
of the subject fusion proteins, an appropriate pharmacological treatment
protocol, e.g., a protocol employing imatinib mesylate is then employed
to treat the patient. Alternatively, where a patient is diagnosed as not
having the subject fusion protein, or the patient is diagnosed as having
the fusion protein but also include a variant of the fusion protein that
is associated with resistance to a particular active agent (e.g., the
T674I mutation) other protocols are then employed.

Additional Utilities

[0222]The subject polypeptide and nucleic acid compositions find use in a
variety of additional applications. Applications in which the subject
polypeptide and nucleic acid compositions find use include: (a) the
identification of homologs; (b) the identification of expression
regulatory factors; (c) as probes and primers in hybridization
applications, e.g. PCR; (d) the identification of expression patterns in
biological specimens; etc.

[0223]A. Identification of Homologs

[0224]Homologs are identified by any of a number of methods. A fragment of
the provided cDNA may be used as a hybridization probe against a cDNA
library from the target organism of interest, where low stringency
conditions are used. The probe may be a large fragment, or one or more
short degenerate primers. Nucleic acids having sequence similarity are
detected by hybridization under low stringency conditions, for example,
at 50° C. and 6×SSC (0.9 M sodium chloride/0.09 M sodium
citrate) and remain bound when subjected to washing at 55° C. in
1×SSC (0.15 M sodium chloride/0.015 M sodium citrate). Sequence
identity may be determined by hybridization under stringent conditions,
for example, at 50° C. or higher and 0.1×SSC (15 mM sodium
chloride/01.5 mM sodium citrate). Nucleic acids having a region of
substantial identity to the provided sequences, e.g. allelic variants,
genetically altered versions of the gene, etc., bind to the provided
sequences under stringent hybridization conditions. By using probes,
particularly labeled probes of DNA sequences, one can isolate homologous
or related genes.

[0225]B. Identification of Expression Regulatory Factors

[0226]Alternatively, mutations may be introduced into the promoter region
to determine the effect of altering expression in experimentally defined
systems. Methods for the identification of specific DNA motifs involved
in the binding of transcriptional factors are known in the art, e.g.
sequence similarity to known binding motifs, gel retardation studies,
etc. For examples, see Blackwell et al., (1995), Mol. Med. 1:194-205;
Mortlock et al. (1996), Genome Res. 6:327-33; and Joulin and Richard-Foy
(1995), Eur. J. Biochem. 232:620-626.

[0227]The regulatory sequences may be used to identify cis acting
sequences required for transcriptional or translational regulation of
gene expression, especially in different tissues or stages of
development, and to identify cis acting sequences and trans-acting
factors that regulate or mediate gene expression. Such transcription or
translational control regions may be operably linked to a fusion protein
gene in order to promote expression of wild type or altered or other
proteins of interest in cultured cells, or in embryonic, fetal or adult
tissues, and for gene therapy.

[0228]C. Probes and Primers

[0229]Small DNA fragments are useful as primers for PCR, hybridization
screening probes, etc. Larger DNA fragments, i.e. greater than 100 nt are
useful for production of the encoded polypeptide, as described in the
previous section. For use in amplification reactions, such as PCR, a pair
of primers will be used. The exact composition of the primer sequences is
not critical to the invention, but for most applications the primers will
hybridize to the subject sequence under stringent conditions, as known in
the art. It is preferable to choose a pair of primers that will generate
an amplification product of at least about 50 nt, preferably at least
about 100 nt. Algorithms for the selection of primer sequences are
generally known, and are available in commercial software packages.
Amplification primers hybridize to complementary strands of DNA, and will
prime extension towards each other.

[0231]The DNA may also be used to identify expression of the gene in a
biological specimen. The manner in which one probes cells for the
presence of particular nucleotide sequences, as genomic DNA or RNA, is
well established in the literature. Briefly, DNA or mRNA is isolated from
a cell sample. The mRNA may be amplified by RT-PCR, using reverse
transcriptase to form a complementary DNA strand, followed by polymerase
chain reaction amplification using primers specific for the subject DNA
sequences. Alternatively, the mRNA sample is separated by gel
electrophoresis, transferred to a suitable support, e.g. nitrocellulose,
nylon, etc., and then probed with a fragment of the subject DNA as a
probe. Other techniques, such as oligonucleotide ligation assays, in situ
hybridizations, and hybridization to DNA probes arrayed on a solid chip
may also find use. Detection of mRNA hybridizing to the subject sequence
is indicative of gene expression in the sample.

[0232]E. Preparation of Mutants

[0233]The sequence of a gene, including flanking promoter regions and
coding regions, may be mutated in various ways known in the art to
generate targeted changes in promoter strength, sequence of the encoded
protein, etc. The DNA sequence or protein product of such a mutation will
usually be substantially similar to the sequences provided herein, i.e.
will differ by at least one nucleotide or amino acid, respectively, and
may differ by at least two or more, e.g., 5, 10, 20 or more nucleotides
or amino acids. The sequence changes may be substitutions, insertions,
deletions, or a combination thereof. Deletions may further include larger
changes, such as deletions of a domain or exon. Other modifications of
interest include epitope tagging, e.g. with the FLAG system, HA, etc. For
studies of subcellular localization, fusion proteins with green
fluorescent proteins (GFP) may be used.

[0235]The following examples are offered by way of illustration and not by
way of limitation.

EXAMPLES

Materials and Methods

[0236]A. Compounds

[0237]Imatinib mesylate was extracted from capsules of Gleevec®.
Vatalanib was prepared according to the published procedure (Bold et al.,
J. Med. Chem. (2000) 43:2310-2323. THRX-165724 was prepared by coupling
piperazine to the carboxyl group of SU6668 (Sun et al., J. Med. Chem.
(1999) 42:5120-5130) as described in Example 1a of U.S. patent
application Ser. No. 10/327,385.

[0241]The EOL-1 and the BaF3 cell lines were obtained from the DSMZ
(Braunschweig, Germany). The basic culture medium for the EOL-1 and BaF3
cell lines was RPMI1640 (Gibco-BRL) supplemented with 10% FBS, 100 U/ml
penicillin and 100 U/ml streptomycin. The medium for BaF3 cells was also
supplemented with 1 ng/ml IL-3 (Biosource International). A BaF3 cell
line expressing NM--030917-PDGFRα was created by
electroporation of BaF3 cells at 300 mV/960 After electroporation, the
BaF3 cells were maintained in IL-3 containing medium for 48 h, selected
in IL-3 containing medium plus 1 mg/ml G418 for 10 days, and subcloned by
limiting dilution.

[0242]D. Cell Viability Assays

[0243]Cell viability was assessed by tetrazolium salt reduction using the
MTT assay (Roche). In a 96-well plate, 5×104 cells/well were
plated in the presence of serial dilutions of compounds. The cells were
incubated for 72 h prior to addition of MTT substrate.

[0244]E. Immunoprecipitation and Western Blotting

[0245]Antibodies against PDGFRα/β and against phosphotyrosine
(4G10) were purchased from Upstate Biotechnology. For each
immunoprecipitation, 1×107 cells were lysed in 0.75 ml
modified RIPA buffer (50 mM Tris-HCl pH 7.4, 1% NP-40, 150 mM NaCl, 1 mM
EDTA, 1 mM Na3VO4, Protease inhibitor cocktail (Roche)). The
lysates were incubated with the appropriate antibody and Protein G beads
(Sigma) overnight at 4° C. The immunocomplexes were recovered by
centrifugation, washed with RIPA buffer, boiled in sample buffer and
resolved by SDS-PAGE. The proteins were transferred to a PVDF membrane
(Invitrogen), blocked with PBS/0.1% Tween/3% BSA and probed with a
specific antibody for 3 h at room temperature. Subsequently, the blots
were washed with PBS/0.1% Tween. Specific antibody binding was detected
with a horseradish-peroxidase coupled secondary antibody followed by
enhanced chemiluminescence ECL (Amersham) and exposure to film. The
primary antibody was typically stripped with ImmunoPure IgG Elution
Buffer (Pierce) for re-probing of the blot with a second antibody.

[0246]F. Phosphorylation Inhibition Assay

[0247]Cells (1×107) were incubated in 3 ml media with the
indicated concentration of drug for 1 h. The cells were subsequently
lysed and immunoprecipitated with the appropriate antibody. Then,
SDS-PAGE was performed followed by immunoblotting with the
anti-phosphotyrosine antibody 4G10.

[0248]G. Protein Digestion and Peptide Analysis

[0249]This work was performed by Proteomic Research Services, Inc. (PRS,
Ann Arbor, Mich.). The sample was provided to PRS in the form of 50 μl
of protein G immunoaffinity resin to which was bound tyrosine
phosphoproteins from 1×108 EOL-1 cells via antibody 4G10. The
proteins were fractionated by SDS-PAGE and visualized by staining with
SYPRO ruby. Plugs were chosen for excision based on an overlay of the
SYPRO-stained lane with that of a companion lane visualized by Western
blotting with 4G10. The plugs were subjected to in-gel digestion with
trypsin (ProGest) and a portion of the supernatant was used for analysis
by matrix-assisted laser desorption-ionization mass spectrometry
(MALDI/MS). MALDI/MS data were acquired on an Applied Biosystems Voyager
DE-STR instrument and the observed m/z values were submitted to a search
for peptide mass fingerprints by the software package ProFound from
Proteometrics, querying the NCBInr database. In cases where MALDI/MS
analysis was inconclusive, samples were analyzed by nano liquid
chromatography followed by 2-dimensional mass spectrometry (LC/MS/MS) on
a Micromass Q-T of2 instrument. The MS/MS data were searched using the
search engine Mascot from Matrix Science (www.matrixscience.com).

Example 1

Discovery of a Novel Oncokinase in Hypereosinophilic Syndrome

[0250]Imatinib mesylate (STI-571/Novartis or Gleevec®) (hereinafter
"imatinib") was tested against various CML and AML cell lines.
Surprisingly, it was discovered that imatinib was a potent inducer of
apoptosis in EOL-1 cells. The EOL-1 cell line was established in 1984
from the peripheral blood of a 33-year-old man with acute myeloid
(eosinophilic) leukemia following hypereosinophilic syndrome. The above
discovery was subsequently confirmed by a report that four HES patients
responded well to imatinib therapy. See Schaller et al., Med. Gen. Med.
(Sep. 7, 2001) 3:9; and Lancet (May 4, 2002); 359(9317):1577-8.

[0251]In a 96-well plate, MV4-11, BV173 and EOL-1 cells were incubated
with increasing concentrations of imatinib for 72 hours. Then, using the
MTT assay, the viability of the cells was assessed. The absorbance at 550
nM is a measure for viability. The results are provided graphically in
FIG. 1.

[0252]As can be seen in FIG. 1, imatinib potently modulates the viability
of EOL-1 cells (IC50: ˜100 pM). Specifically, imatinib is a
very potent inhibitor of eosinophilic cell line EOL-1. The CML cell line
BV173 also shows reduced viability in the presence of imatinib but the
drug is considerably less potent against this cell line (IC50: 250
nM). Even at 10 μM, imatinib has very little effect on the viability
of MV4-11 cells. The MV4-11 cell line is an acute myeloid
(myelomonocytic) leukemia cell line.

[0253]Imatinib was expected to reduce the viability of the CML cell line
BV173 since these cells express Bcr-Abl. MV4-11 cells do not express
Bcr-Abl and as a result they are not sensitive to the drug. The high
sensitivity of EOL-1 cells towards imatinib was surprising and
unexpected. Like cells from most HES patients, EOL-1 cells are known to
have no chromosomal translocations. Hence, the sensitivity of these cells
towards imatinib cannot be based on the inhibition of Bcr-Abl.
Furthermore, imatinib is more than 100-fold more potent against EOL-1
than against BV173. Other CML cell lines were tested against imatinib and
all of them showed IC50s similar to the one obtained for BV173. The
lack of the typical CML chromosomal translocation and the extraordinary
sensitivity of EOL-1 towards imatinib indicated that imatinib was
inhibiting one or more novel targets in these cells.

[0254]In order to identify the novel imatinib target in EOL-1, the
phosphoprotein profile in EOL-1 cells was first observed. EOL-1 cells
were left untreated or were treated with increasing concentrations of
imatinib for 2 hours. The cells were subsequently lysed and
immunoprecipitated with an antibody against phosphotyrosine (4G10,
obtained from Upstate Biotechnology). Subsequently, a Western Blot
analysis was performed using the same anti-phosphotyrosine antibody.

[0255]The results demonstrated that, in EOL-1, there is a prominent
phosphoprotein of 110 kDa molecular weight. This 110 kDa phosphoprotein
is not phosphorylated in the presence of imatinib (at about 100 nM).

[0256]The 110 kDa phosphoprotein was predicted to be an activated kinase
that is inhibited by imatinib. To further characterize the protein, a
slice containing the 110 kDa molecular weight region was cut from a gel
containing immunoprecipitated EOL-1 phosphoproteins. The proteins in the
gel slice were digested with the protease trypsin and the identities of
the tryptic peptide fragments were determined using mass spectroscopy.
Three proteins were identified: [0257]1. Nucleolin. Nucleolin is an
abundant, 105 kDa phosphoprotein involved in the assembly of ribosomes.
Based on these known features, Nucleolin was determined not to be the
likely target for imatinib. [0258]2. NM--030917 gene product. This
gene is expressed in many tissues but its function is not known. However,
it is not a kinase and therefore is unlikely to be a direct target for
imatinib. All the NM--030917 gene product peptides mapped to the
N-terminus of the protein. [0259]3. PDGFRα. PDGFRα is a
receptor tyrosine kinase. Imatinib is known to inhibit the closely
related PDGFRβ with an IC50 of 300-1000 nM. Therefore,
PDGFRα was identified as a potential candidate for the imatinib
target in EOL-1. Surprisingly, all the PDGFRα peptides identified
mapped into the C-terminus of the protein. The C-terminus contains the
kinase domain of the receptor.

[0260]Accordingly, the following experiment was performed to verify that
the 110 kDa phosphoprotein contains the C-terminus of the PDGFRα
receptor.

[0261]EOL-1 cells were left untreated or were treated with increasing
concentrations of imatinib for 2 hours. The cells were lysed and the
lysates were immunoprecipitated with an anti-PDGFRα antibody that
recognizes an epitope in the C-terminus of the receptor. Subsequently, a
Western Blot analysis was performed using the anti-phosphotyrosine
antibody.

[0262]It was found that the anti-phosphotyrosine antibody
immunoprecipitated a phosphoprotein of 110 kDa. This phosphoprotein was
dephosphorylated in the presence of imatinib. The IC50 was ˜30
nM. This data suggested that the 110 kDa phosphoprotein in EOL-1 contains
the C-terminus of PDGFRα.

[0263]PDGFRα is a receptor with a molecular weight of 185 kDa in its
wild-type form. The fact that the C-terminus of PDGFRα was clearly
present in the 110 kDa phosphoprotein meant that the receptor was
mutated. Such a mutation could also explain why the kinase domain of the
receptor was constitutively activated. Activated tyrosine kinases play a
role in many cancers. Often, the activation is a result of a mutation in
the kinase. There are two principal sets of mechanisms by which tyrosine
kinases are found to be activated by mutation. The first mechanisms
include point mutations, deletions or small duplications in the gene
encoding the kinase. The second mechanisms include the formation of
fusion proteins like Bcr-Abl which are usually a result of chromosomal
translocation.

[0264]The above findings supported a conclusion that there was a fusion
between the PDGFRα kinase domain and a second protein, analogous to
Bcr-Abl. It is known that there are no gross chromosomal abnormalities in
EOL-1 cells. There is a small deletion on chromosome 9 but the
PDGFRα resides on chromosome 4. If PDGFRα was fused to
another gene, that gene had to be nearby on chromosome 4 in order not to
be cytogenetically obvious. NM--030917, the gene product of which
was found to be present in the same gel slice as PDGFRα, is located
in close proximity to the PDGFRα gene on chromosome 4. Thus, a
small rearrangement on chromosome 4 would lead to the fusion of these two
genes.

[0265]To test this possibility, RNA from EOL-1 cells was isolated and used
to generate cDNA with a primer that primes in the non-coding, 3' region
of the PDGFRα gene. The cDNA was used as a template for a PCR
reaction with a primer pair priming at the very 3' end of the
PDGFRα and at the very 5' end of NM--030917. The PCR yielded a
fragment of about 2.5 kb that was cloned. Sequencing of various clones
revealed the following: [0266]1. NM--030917 and PDGFRα form
a fusion transcript in which the intracellular domain of PDGFRα
containing the kinase domain is fused to the N-terminus of
NM--030917. [0267]2. The fusion is in frame giving rise to an open
reading sequence comprising 2502 base pairs corresponding to 834 amino
acids. [0268]3. The NM--030917 fragment is alternatively spliced
resulting in the optional addition of one or two exons. [0269]4. A
tryptic peptide encompassing amino acid sequence from the predicted
NM--030917 and PDGFRα fusion point was identified in the EOL-1
110 kDa phosphoprotein gel slice. Hence, the fusion protein is expressed
in EOL-1 cells.

[0270]The coding sequence for the NM--030917-PDGFRα fusion gene
is as follows:

The underlined sequences are alternatively spliced. None or one or both
sequences may be present in a given transcript. These additional
alternatives are provided as SEQ ID NOS: 06, 07 and 08 in the attached
Sequence Listing.

The underlined peptides are alternatively spliced sequences. None or one
or both sequences may be present in a given fusion protein. These
alternative sequences are provided as SEQ ID NOS: 02, 03 and 04 in the
attached sequence listing.Double Underline: Peptide identified by mass
spec which contains sequences from NM--030917 and PDGFRα. (The
fusion is between Q (NM--030917) and L (PDGFRα)

[0272]Primer pairs flanking the fused exons of NM--030917 and
PDGFRα on the normal chromosome 4 were designed. Using genomic DNA,
those exons in EOL-1 cells as well as other leukemia cell lines were
amplified using the designed primer pairs in a PCR protocol. When the 5'
primer of the fused NM--030917 exon was combined with the 3' primer
of the fused PDGFRα exon, a fragment of 1100 base pairs from EOL-1
genomic DNA but not from any other cell line tested was obtained in the
PCR protocol. This fragment contained the genomic recombination point and
it is derived from the mutant chromosome 4. The 1100 base pair fragment
was sequenced to characterize the genomic recombination point. The
following observations were made: [0273]1. The recombination point
deletes approximately one million base pairs on chromosome 4. This leads
to the fusion of an intron in NM--030917 to the middle of exon 12 in
PDGFRα. [0274]2. The splice donor dinucleotide GT in the
NM--030917 intron recognizes the first AG dinucleotide in the PDGFR
alpha exon as the splice acceptor site. The splicing reaction results in
a fusion that maintains the reading frame of the PDGFR gene.

The underlined bases denote the splice donor and splice acceptor sites of
the intron that comprise the fusion point.

[0276]In summary, eosinophilia cells in patients have been shown to
undergo apoptosis when exposed to imatinib. The eosinophilic AML cell
line EOL-1 has been shown above to be similarly sensitive to imatinib and
therefore represents a good model system to identify targets for imatinib
in this disease. The EOL-1 cells were discovered to have a chromosomal
rearrangement on chromosome 4 which leads to the expression of a fusion
protein consisting of an uncharacterized protein and the cytoplasmic
domain of PDGFRα. The fusion protein is highly phosphorylated in
EOL-1 cells which is a reflection of an activated state of the
PDGFRα kinase domain. The phosphorylation of the fusion protein can
be inhibited with imatinib. The IC50 is ˜30 nM. The above data
show that the kinase domain of PDGFRα expressed as a fusion protein
is a target for imatinib in EOL-1.

[0277]Thus, the above results show that PDGFRα fusion proteins play
an important role in hypereosinophilic syndrome and acute myeloid
(eosinophilic) leukemia, analogous to Bcr-Abl in CML. The fusion proteins
may differ in the exact genomic location where NM--030917 and
PDGFRα are recombined.

[0278]Furthermore, since fusion with NM--030917 is able to activate
the kinase domain of PDGFRα, then fusion of an N-terminal domain of
the NM--030917 protein activates other kinase domains in certain
embodiments. For example, just downstream from PDGFRα on chromosome
4 are two more receptor tyrosine kinases, namely c-kit and VEGFR-2. As
such, in other hyperproliferative diseases, fusions of NM--030917
and c-kit or VEGFR-2 are expected to be present.

Example 2

Inhibition of Cellular N M--030917-PDGFRα Autophosphorylation

[0279]Using Western Blot analysis, the potency of THRX-165724 (described
in Example 1 of patent application Ser. No. 10/327,385) and vatalanib
(PTK787) (described in Examples 1 to 4 of U.S. Pat. No. 6,258,812 B1) in
inhibiting cellular NM--030917-PDGFRα autophosphorylation was
assessed. It was found that both of these kinase inhibitors have activity
in this assay. In parallel, both inhibitors were tested for their ability
to induce apoptosis in EOL-1 cells. The IC50s obtained in the two
assays are listed below:

[0280]The IC50s for THRX-165724 and vatalanib (PTK787) correlate well
between the two assays. The above results demonstrate that THRX-165724
and vatalanib (PTK787) at 10-30 and 100-300 nM, respectively, induce
apoptosis by inhibiting NM--030917-PDGFRα.

Example 3

Cell-Transforming Potential of NM--030917-PDGFRα

[0281]Mutationally activated tyrosine kinases, as found in many cancers,
can transform the murine myeloid cell line BaF3 to interleukin-3
independence. In order to determine if NM--030917-PDGFRα has
the ability to transform cells, a BaF3 cell line was established. BaF3
cells expressing NM--030917-PDGFRα from EOL-1 were found to be
IL-3 independent. The fusion protein in these cells was constitutively
phosphorylated and the phosphorylation was inhibited by imatinib with an
IC50 of 30 nM, the same value as obtained in EOL-1. Inhibition with
imatinib, vatalanib and THRX-165724 resulted in reduced viability of the
BaF3 NM--030917-PDGFRα cells with IC50s similar to the
potency of the drugs in EOL-1. The effect of the inhibitors was overcome
in the presence of IL-3. NM--030917-PDGFRα is also likely to
be the target for imatinib, vatalanib and THRX-165724 in EOL-1 since the
expression of the fusion gene conferred IL-3 independent growth to BaF3
cells which was inhibited by the three drugs at concentrations similar to
those at which they inhibited the viability of EOL-1 cells. The viability
of these BaF3 cells in the presence of the PDGFRα inhibitors could
be maintained by exogenous IL-3.

Example 4

Identification of NM--030917-PDGFRα in HES Patient Cells

[0282]In order to determine if the NM--030917-PDGFRα fusion was
present in HES patients, blood cells from four patients diagnosed with
HES were obtained. Patients 1 and 2 had been treated with imatinib.
Patient 1 responded to treatment, but patient 2 did not. After showing a
complete hematologic remission, patient 1 relapsed and died. This patient
had multiple clonal cytogenetic abnormalities which led to the diagnosis
of CEL. Genomic DNA as well as total RNA and cDNA were prepared from all
patient cells except for patient 1 for whom only genomic DNA, but no RNA
and cDNA were obtained from cells before imatinib treatment. The cDNA
samples were subjected to PCR with a primer pair spanning the fusion
point determined in EOL-1 cells. In the samples from patients 1 and 3,
fragments could be amplified from the cDNA that constituted in-frame
fusion transcripts between NM--030917 and PDGFRα (FIG. 3A). No
NM--030917-PDGFRα fusion was detected in patients 2 and 4. In
patient 1, the fusion transcript connects exon 8 of NM--030917
within exon 12 of PDGFRα. A similar approach as for EOL-1 was used
to identify the genomic breakpoint. In patient 1, the intronic break is
at an AG dinucleotide that serves as the splice acceptor site so that
exon 8 in NM--030917 and part of exon 12 in PDGFRα are fused
in-frame in the fusion transcript (FIG. 3B). The
NM--030917-PDGFRα fusion in patient 1 was detected in genomic
DNA preparations derived from cells taken before the start of imatinib
therapy and at the time of relapse. The analysis of
NM--030917-PDGFRα cDNA at the time of relapse revealed a point
mutation in the PDGFRα kinase domain. The mutation affects amino
acid position 674 in PDGFRα resulting in the substitution of
threonine by isoleucine (T6741).

[0283]In patient 3, the fusion transcript as well as the genomic break are
identical to the mutation found in EOL-1 cells (FIGS. 3A and 3B).

[0284]Amplification and sequencing of genomic DNA from EOL-1 cells
revealed that the genomic breakpoint junctions fell within an intron
following exon 11 of NM--030917 and within exon 12 of PDGFRα.
The same mutation was observed in patient 3 and a similar submicroscopic
deletion was discovered in patient 1. In patient 1 the resulting
transcript fused a different site in the NM--030917 gene to a
distinct site in PDGFRα exon 12. Exon 12 encompasses the
cytoplasmic juxtamembrane region of PDGFRα, followed by the kinase
domain.

[0285]Patient 1 relapsed and died after initially having shown a complete
remission in response to imatinib. At the time of relapse, this patient
had a T674I mutation in the PDGFRα kinase domain. T674 in
PDGFRα corresponds to T315 in c-Abl. Based on the crystal structure
of the catalytic domain of c-Abl bound to a derivative of imatinib, T315
forms part of the imatinib binding pocket and establishes a hydrogen bond
with the drug (Schindler et al., Science (2000) 289:1938-1942). The T315I
mutation ablates the kinase inhibitory activity of imatinib for Bcr-Abl
and it is one of the most common mutations found in CML patients who are
resistant to the drug (Gorre et al., Science (2001) 293:876-880; Branford
et al., Blood (2002) 99:3472-3475). The above indicates that that the
T674I mutation underlies the relapse of patient 1 and therefore provides
further evidence that NM--030917-PDGFRα is the target of
imatinib.

[0286]Two reports describe patients with myeloproliferative disorders with
eosinophilia who have chromosomal translocations at 4q11-12 (Duell et
al., Cancer Genet. Cytogenet. (1997) 94:91-94; Schoffski et al., Ann.
Hematol. (2000) 79:95-98). These translocations may involve either
NM--030917 or PDGFRα leading to different disease promoting
fusion proteins. For example, NM--030917 may play a role analogous
to that played by Tel or Bcr--promoting dimerization and, thus, the
activation of known oncogenic fusion kinases. Like PDGFRα, the
c-Kit gene is located on chromosome 4q12. Submicroscopic deletions could
also result in NM--030917-c-Kit fusions. A search of the NCBI EST
database reveals that NM--030917 is expressed in many tissues and
organs suggesting that NM--030917-fusion kinases may not be
restricted to cells of hematological origin. The protein encoded by
NM--030917 is homologous (26% over 307 residues) to yeast protein
FIP1, a component of a polyadenylation factor (Preker et al., Cell (1995)
81:379-389).

[0288]In summary, the above experiments demonstrate that the novel
NM--030917-PDGFRα genomic rearrangement, discovered in the
eosinophilic EOL-1 cell line, is present in a subset of patients
diagnosed with HES. Cell viability and phosphorylation data show that the
NM--030917-PDGFRα kinase encoded by the novel fusion gene
plays a central role in the disease process of these HES patients.
Accordingly, HES in which the NM--030917-PDGFRα fusion is
detected may be described as chronic eosinophilic leukemia.

[0289]It is evident from the above results and discussion that the subject
invention provides for important new targets for the treatment of various
disease conditions, including proliferative diseases, such as cancer. In
addition to providing the subject targets, the invention further provides
important new methods of diagnosis and treatment, which will provide
significant benefits the medical and related fields. Accordingly, the
subject invention represents a significant contribution to the art.

[0290]All publications and patents cited in this specification are herein
incorporated by reference as if each individual publication or patent
were specifically and individually indicated to be incorporated by
reference. The citation of any publication is for its disclosure prior to
the filing date and should not be construed as an admission that the
present invention is not entitled to antedate such publication by virtue
of prior invention.

[0291]Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity of
understanding, it is readily apparent to those of ordinary skill in the
art in light of the teachings of this invention that certain changes and
modifications may be made thereto without departing from the spirit or
scope of the appended claims.